Codon optimized gla genes and uses thereof

ABSTRACT

The present disclosure provides codon optimized nucleotide sequences encoding hum n alpha-galactosidase A, vectors, and host cells comprising codon optimized alpha-galactosidase A sequences, and methods of treating disorders such as Fabry disease comprising administering to the subject a codon optimized sequence encoding human alpha-galactosidase A.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.18/045,650, filed Oct. 11, 2022, which is a continuation ofinternational patent application number PCT/US2021/029146, fled Apr. 26,2021, which claims the benefit of U.S. Provisional Patent ApplicationNos. 63/114,195, filed Nov. 16, 2020 and 63/016,207, filed Apr. 27,2020, the entire contents of each of which is incorporated herein byreference.

SEQUENCE LISTING SUBMISSION VIA EFS-WEB

A computer readable XML file, entitled “090400-5014-US02-Sequence-Listing” created on or about Sep. 8, 2023, with a file sizeof about 20,000 bytes, contains the sequence listing for thisapplication and is hereby incorporated by reference in its entirety

BACKGROUND OF THE INVENTION

Fabry disease is an X-linked disorder in which mutations in the GLA gene(encoding α-galactosidase A, AGA) result in reduced or absent AGA enzymeactivity and consequent accumulation of globotriaosylceramide (Gb3). Gb3is considered cytotoxic to cardiomyocytes and endothelial cells(kidney/heart/neuron) resulting in significant morbidity and shortenedlife expectancy. Administration of recombinant AGA (ERT) appears to slowdisease progression in some tissues. Likely due to poor uptake intocells, for example in the heart, there remains significant unmet medicalneed. In addition, cardiovascular disease remains the mot common causeof mortality in Fabry disease (75% of all known deaths). Thus, there isa compelling need for a durable treatment such as a singleadministration intravenous gene therapeutic targeted to key tissues thatexpress GLA cell-autonomously, reducing Gb3 and thereby improvingclinical outcomes.

One impediment with respect to cardiac gene therapy is insufficienttransduction of human cardiomyocytes following intravenousadministration. Current AAV vectors in development traffic predominantlyto the liver and are not targeted to heart tissue. Wild-type AAV1 hasbeen evaluated clinically, but ultimately did not result in animprovement in the primary endpoint of recurrent heart failure eventscompared to placebo. Novel heart targeted AAV variants may provideeffective treatment of Fabry disease cardiomyopathy.

SUMMARY OF THE INVENTION

Disclosed are codon optimized nucleic acid molecules encoding a humangalactosidase A (AGA) protein. In one aspect, the disclosure provides anucleic acid comprising the nucleotide sequence of SEQ ID NO:1 or anucleic acid comprising a nucleotide sequence at least 90%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% identicalto the nucleotide sequence of SEQ ID NO:1 and which encodes a human AGApolypeptide having the amino acid sequence of SEQ ID NO:2. In someembodiments, a nucleic acid comprising or consisting of the nucleotidesequence of SEQ ID NO:1 is provided. In related embodiments, the nucleicacid is expressed at a higher level compared with the level ofexpression of a wild type GLA nucleic acid sequence (e.g. SEQ ID NO:3)in an otherwise identical cell. SEQ ID NO:3 (consensus CDS sequence no.CCDS14484.1; www.uniprot.org/uniprot/Q7X4P3) is set forth below:

(SEQ ID NO: 3) ATGCAGCTGAGGAACCCAGAACTACATCTGGGCTGCGCGCTTGCGCTTCGCTTCCTGGCCCTCGTTTCCTGGGACATCCCTGGGGCTAGAGCACTGGACAATGGATTGGCAAGGACGCCTACCATGGGCTGGCTGCACTGGGAGCGCTTCATGTGCAACCTTGACTGCCAGGAAGAGCCAGATTCCTGCATCAGTGAGAAGCTCTTCATGGAGATGGCAGAGCTCATGGTCTCAGAAGGCTGGAAGGATGCAGGTTATGAGTACCTCTGCATTGATGACTGTTGGATGGCTCCCCAAAGAGATTCAGAAGGCAGACTTCAGGCAGACCCTCAGCGCTTTCCTCATGGGATTCGCCAGCTAGCTAATTATGTTCACAGCAAAGGACTGAAGCTAGGGATTTATGCAGATGTTGGAAATAAAACCTGCGCAGGCTTCCCTGGGAGTTTTGGATACTACGACATTGATGCCCAGACCTTTGCTGACTGGGGAGTAGATCTGCTAAAATTTGATGGTTGTTACTGTGACAGTTTGGAAAATTTGGCAGATGGTTATAAGCACATGTCCTTGGCCCTGAATAGGACTGGCAGAAGCATTGTGTACTCCTGTGAGTGGCCTCTTTATATGTGGCCCTTTCAAAAGCCCAATTATACAGAAATCCGACAGTACTGCAATCACTGGCGAAATTTTGCTGACATTGATGATTCCTGGAAAAGTATAAAGAGTATCTTGGACTGGACATCTTTTAACCAGGAGAGAATTGTTGATGTTGCTGGACCAGGGGGTTGGAATGACCCAGATATGTTAGTGATTGGCAACTTTGGCCTCAGCTGGAATCAGCAAGTAACTCAGATGGCCCTCTGGGCTATCATGGCTGCTCCTTTATTCATGTCTAATGACCTCCGACACATCAGCCCTCAAGCCAAAGCTCTCCTTCAGGATAAGGACGTAATTGCCATCAATCAGGACCCCTTGGGCAAGCAAGGGTACCAGCTTAGACAGGGAGACAACTTTGAAGTGTGGGAACGACCTCTCTCAGGCTTAGCCTGGGCTGTAGCTATGATAAACCGGCAGGAGATTGGTGGACCTCGCTCTTATACCATCGCAGTTGCTTCCCTGGGTAAAGGAGTGGCCTGTAATCCTGCCTGCTTCATCACACAGCTCCTCCCTGTGAAAAGGAAGCTAGGGTTCTATGAATGGACTTCAAGGTTAAGAAGTCACATAAATCCCACAGGCACTGTTTTGCTTCAGCTAGAAAATACAATGCAGATGTCATTAAAAGACTTACTTTAA

In some aspects, a codon optimized nucleic acid molecule as hereindescribed has a human codon adaptation index that is increased relativeto that of the wild type GLA cDNA (GenBank Accession No. NM_000169.3;SEQ ID NO:3). In some embodiments, the codon optimized nucleic acidmolecule has a human codon adaptation index of at least about 0.80, atleast about 0.83, at least about 0.85, at least about 0.88, at leastabout 0.90, at least about 0.92 or at least about 0.93.

In certain embodiments, the nucleic acid contains a higher percentage ofG/C nucleotides compared to the percentage of G/C nucleotides in SEQ IDNO:3. In other embodiments, the nucleic acid contains a percentage ofG/C nucleotides that is at least about 49%, at least about 51%, at leastabout 53%, at least about 55%, at least about 57%, at least about 57.9%or is about 57.9% In related embodiments, the nucleic acid contains apercentage of G/C nucleotides that is between about 49% and 60%, betweenabout 50% and 59%, between about 55% and 9% or between about 57% andabout 59%.

In other embodiments, the nucleic acid comprises one or more optimizedparameters relative to SEQ ID NO:3: frequency of optimal codons;reduction in maximum length of direct repeat sequences; removal ofrestriction enzymes, removal of CIS-acting elements, and removal ofdestabilizing elements. In another embodiment, the nucleic acid isoperatively linked to at least one transcription control sequence,preferably a transcription control sequence that is heterologous to thenucleic acid. In some aspects, the transcription control sequence is acell- or tissue-specific promoter that results in cell-specificexpression of the nucleic acid e.g. in cardiac or skeletal muscle cells.In other aspects, the transcription control sequence is a constitutivepromoter that results m similar expression level of the nucleic acid inmany cell types (e.g. a CAG, CBA (chicken beta actin) or CMV promoter).In preferred embodiments, the transcription control sequence comprises aCAG promoter comprising (i) the cytomegalovirus (CMV) early enhancerelement, (ii) the promoter, first exon and first intron of chickenbeta-actin gene and (iii) the splice acceptor of the rabbit beta-globingene as described in Miyazaki et al., Gene 79(2): 269-77 (1989). In aparticularly preferred embodiment, the CAG promoter comprises thesequence of SEQ ID NQ:5 or comprises a sequence at least 95%, at least%%, at least 97%, at least 98% or at least 99% identical thereto.

(SEQ ID NO: 5) ACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTGCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTGCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGGGGGGGGGGCGAGGCGGAGAGGTGCGGGGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGGGGGGAGTCGCTGCGACGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGGGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGGGGTGCCCCGCGGTGCGGGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGGGCGTCGGTCGGGCTGCAACCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGGGGGGGGGGCGGCAGGTGGGGGTGCCGGGCGGGGGGGGGCGGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGTGGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGGGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGGGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGGGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTC TTTTTCCTACAG

In related embodiments, provided herein is an expression cassettecomprising a nucleic acid comprising the nucleotide sequence of SEQ IDNO:1, or a nucleotide sequence at least 90% identical thereto, operablylinked to an expression control sequence.

In related embodiments, provided herein is a vector comprising a nucleicacid comprising the nucleotide sequence of SEQ ID NO:1, or a nucleotidesequence at least 90% or at least 95% identical thereto. In preferredembodiments, the vector is a recombinant adeno-associated (rAAV)expression vector. In some embodiments, the rAAV vector comprises anative capsid (e.g. a capsid of AAV serotype 1, AAV serotype 2, AAVserotype 6, or AAV serotype 8). In other embodiments, the rAAV vectorcomprises a capsid that is modified (e.g. comprises one or more peptideinsertions and/or one or more amino acid substitutions (e.g. tyrosine tophenylalanine) and/or amino acid insertions or amino acid deletions)relative to a native AAV capsid (e.g. comprising one or moremodifications relative to an AAV capsid of serotype 1, 2, 6 or 8). In aparticularly preferred embodiment, the rAAV vector comprises a capsidcomprising a capsid protein of SEQ ID NO:4 or a sequence at least 90%,at least 95% or at least 98% identical thereto.

In another embodiment, provided herein is a host cell comprising anucleic acid comprising the nucleotide sequence of SEQ ID NO:1, or anucleotide sequence at least 90% identical thereto. In some aspects, thehost cell is a mammalian cell, including without limitation, a CHO cell,an HEK293 cell, and HEK293T cells, a HeLa cell, a BHK21 cell, a Verocell or a V27 cell. In other aspects, the host cell is a cardiac orskeletal muscle cell (e.g. myoblast, skeletal muscle fibroblast,skeletal muscle satellite cell, cardiomyocyte, cardiac fibroblast,cardiac progenitor cell, smooth muscle cell endothelial and/or diaphragmmuscle cell). In related embodiments, the disclosure provides a methodof increasing expression of a polypeptide of SEQ ID NO: 2 comprisingculturing the host cell under conditions whereby a polypeptide of SEQ IDNO: 2 is expressed by the nucleic acid molecule, wherein the expressionof the polypeptide is increased relative to a host cell cultured underthe same conditions comprising a reference nucleic acid comprising thenucleotide sequence of SEQ ID NO:3 (comparator sequence).

In another embodiment, the disclosure provides a method of increasingexpression of a polypeptide of SEQ ID NO: 2 in a human subjectcomprising administering to the subject an isolated nucleic acidmolecule comprising a nucleotide sequence at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to the nucleotide sequence of SEQ ID NO:1 and which encodes apolypeptide having the amino acid sequence of SEQ ID NO:2 or a vectorcomprising such a nucleotide sequence, wherein the expression of thepolypeptide is increased relative to a reference nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO:3 (comparator sequence)or a vector comprising the reference nucleic acid molecule.

In some embodiments, the disclosure provides a method of treating adisorder associated with insufficient GLA activity in a human subjectcomprising administering to the subject a nucleic acid molecule or avector disclosed herein. In some embodiments, the disorder is Fabrydisease.

DESCRIPTION OF THE DRAWINGS

FIG. 1 AGA Protein Absent from Fabry Disease Fibroblasts. A Western blotanalysis of wildtype and Fabry diseased fibroblasts is shown. Lysates ofFabry fibroblasts lacked AGA protein expression (49 kDa), an indicationof their disease mutation (W162X). Samples were normalized for totalprotein and equally loaded on the gel. Standard protein ladder on thefar left. hrAGA: human recombinant AGA, WT: wildtype, AGA: alphagalactosidase, kDa: kilodaltons.

FIG. 2A-B Fabry Diseased Induced Pluripotent Stem Cell Characterization.Fabry diseased fibroblasts reprogrammed into induced pluripotent stemcells (iPSC) (FIG. 2A) express critical pluripotent transcriptionfactors, Nanog (left, red), Oct 4 (middle, green) and Sox2 (right, red),DAPI (nuclei, blue)(top panel). Fabry iPSC differentiated into all threegerm layers, ectoderm, endoderm and mesoderm and expressed lineagemarkers corresponding to each germ layer, β-tubulin III (left, green),HNF-α (middle, red) and α-smooth muscle actin (left, red), respectively;DAPI (nuclei, blue)(bottom panel). Scale bar=100 μm. HNF4a: bepatocytenuclear factor 4 alpha. Karyotype analysis of Fabry iPSC shows normalhuman male banding and chromosomal arrangement (FIG. 2B).

FIGS. 3A-B Fabry Diseased Induced Pluripotent Stem Cell DerivedCardiomyocyte Characterization. The differentiation of Fabry diseasediPSC into cardiomyocytes, yielded a population of 97% cTNT positivecells when examined by flow cytometry and gated using the IgG control.Scatter plot is a representative plot to show gating scheme (FIG. 3A). Arepresentative image of Fabry iPSC cardiomyocytes followingimmunocytochemistry with cTNT (red) and DAPI (blue), a nuclear stain(FIG. 3B). Scale bar=100 μm. cTNT: cardiac troponin T, cardiomyocytemarker n=3, biologic replicates.

FIGS. 4A-C Transduction of Fabry Diseased iPSC-Cardiomyocytes withrecombinant AAV (rAAV) particles comprising (i) a capsid with a capsidprotein of SEQ ID NO:4 and (ii) a nucleic acid comprising a nucleotidesequence of SEQ ID NO:1 operably linked to a CAG promoter, Leads toExpression of AGA Protein. Immunocytochemistry (ICC) showed expressionof cardiac troponin T (cTnT) (green), alpha-galactosidase (red), andnuclei counterstain with Hoechst (blue) in Fabry iPSC cardiomyocytestransduced with the AAV (FIG. 4A). Fabry iPSC cardiomyocytes transducedwith the AAV were co-stained with cTnT and alpha-galactosidase todetermine the percent of Fabry iPSC cardiomyocytes expressing AGA (FIG.4B). Robust expression of AGA protein seen by Western blot analysis ofFabry iPSC cardiomyocytes after transduction with the AAV (FIG. 4C).AGA: alpha galactosidase. MOI: Multiplicity of infection, NT:non-transduced, Gb3: globotriaosylceramide, n=3, Error bars±StandardDeviation; ***p<0.0001 compared to NT, ††p<0.001, †p<0.01 compared toMOI 25, One-way ANOVA, Tukey post-hoc compared to NT.

FIGS. 5A-5B Enhanced AGA activity following Transduction with rAAVcomprising (i) a capsid with a capsid protein of SEQ ID NO:4 and (ii) anucleic acid comprising a nucleotide sequence of SEQ ID NO:1 operablylinked to a CAG promoter in Fabry Diseased iPSC-Cardiomyocytes. FabryiPSC cardiomyocytes transduced with the AAV showed a dose dependentincrease in AGA activity. Three biologic replicates are quantifiedindividually due to the inherent variability within the kit and platereader for each run. Technical replicates were performed within eachexperiment (FIG. 5A). CC of globotriaosylceramide (Gb3, pink) and nucleicounterstain with Hoechst (blue)(FIG. 5B). AGA=alpha galactosidase,MOI=Multiplicity of infection, NT=non-transduced,Gb3=globotriaosylceramide, n=3, Error bars±Standard Deviation;***p<0.01, ***p<0.004, *p<0.005 compared to NT, ††††p<0.0001,†††p=0.0001, ††p<0.004; †p<0.02 compared to MOI 25, #p<0.02 compared toMOI 100. One-way ANOVA, Tukey post-hoc compared to NT.

FIGS. 6A-6B Characterization of Fabry Diseased Induced Pluripotent StemCell-Derived Endothelial Cells. FIG. 6A: Day 21 of differentiation ofFabry disease iPSC into endothelial cells yielded a population of 99.2%CD31 positive cells and 87.9% CD144 positive when examined by flowcytometry and gated using the IgG control. FIG. 6B: CD31 (green)expression by immunocytochemistry in cultures used in vectorcharacterization. Cells were counterstained with DAPI (blue). Scalebar=100 μM.

FIGS. 7A-7B Transduction of Fabry Disease iPSC-Endothelial Cells withrAAV comprising (i) a capsid with a capsid protein of SEQ ID NO:4 and(ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO:1operably linked to a CAG promoter Leads to Expression of Exogenous AGAby flow and Western. FIG. 7A illustrates flow cytometric expression ofAGA protein in live CD31⁺/CD144⁺ population at four dayspost-transduction in Fabry endothelial cells transduced with the rAAV.FIG. 7B: robust expression of AGA protein seen by western blot analysisof Fabry iPSC-derived endothelial cells after transduction with therAAV. Band densitometry in histogram. AGA=alpha galactosidase.MOI=Multiplicity of infection, NT=non-transduced,Gb3=globotriaosylceramide, n=3, Error bars=Standard Deviation;****p<0.0001, *p<0.05 compared to predecessor MOI, One-way ANOVA, Tukeypost-hoc compared to NT.

FIGS. 8A-8B. Enhanced AGA activity following transduction with rAAVcomprising (i) a capsid with a capsid protein of SEQ ID NO.4 and (ii) anucleic acid comprising a nucleotide sequence of SEQ ID NO:1 operablylinked to a CAG promoter in Fabry Diseased iPSC-Endothelial Cells. FIG.8A: AGA activity was quantified by AGA activity assay at day fourpost-transduction. Fabry iPSC-ECs transduced with the rAAV had more AGAactivity than non-transduced Fabry endothelial cells. FIG. 8B: ICC ofglobotriaosylceramide (Gb3, pink) positive cells and nuclei counterstainwith DAPI (blue). GLA=alpha galactosidase, MOI=Multiplicity ofinfection, NT=non-transduced, Gb3=globotriaosylceramide, n=3, Errorbars=Standard Deviation; *p<0.05 or **p<0.001 compared to preceding MOIs(500, 100, 50, & NT_from top to bottom), One-way ANOVA, Tukey post-hoc.3 experimental and 3 technical replicates each.

FIG. 9A-9B. AGA activity following transduction with rAAV comprising (i)a capsid with a capsid protein of SEQ ID NO:4 and (ii) a nucleic acidcomprising a nucleotide sequence of SEQ ID NO:1 operably linked to a CAGpromoter of SEQ ID NO:5 in a mouse model of Fabry Disease. FIG. 9A: AGAactivity in wildtype and Fabry mouse plasma after a single intravenousdose. FIG. 9I: AGA activity in wild type and Fabry mouse tissues after asingle intravenous dose. Mean±standard deviation; *p<0.01 compared toVehicle-WT, #p<0.01 compared to Vehicle—KO. WT=wildtype mouse, KO=FabryDisease mouse (knockout).

FIG. 10 . Detection of AGA by IHC in Tissues of Normal (WT) or Fabry(KO) Mice 8 Weeks After a Single IV Dose of rAAV comprising (i) a capsidwith a capsid protein of SEQ ID NO:4 and (ii) a nucleic acid comprisinga nucleotide sequence of SEQ ID NO:1 operably linked to a CAG promoterof SEQ ID NO:5. Representative images of four tissues (heart, kidney,liver and small intestine) showing IHC staining for AGA (dark browncolor). IHC staining for each tissue shows a dose-dependent increase inAGA detection after a single intravenous dose of rAAV comprising (i) acapsid with a capsid protein of SEQ ID NO:4 and (ii) a nucleic acidcomprising a nucleotide sequence of SEQ ID NO: 1 operably linked to aCAG promoter of SEQ ID NO:5 in a mouse model of Fabry Disease comparedto wild-type mice treated with vehicle or Fabry mice treated withvehicle. IHC was performed on tissues collected from n=3 animals pergroup.

FIG. 11 . LysoGb3 Levels in Plasma. LysoGb3 reduction in plasma aftersingle IV dose of rAAV comprising (i) a capsid with a capsid protein ofSEQ ID NO:4 and (ii) a nucleic acid comprising a nucleotide sequence ofSEQ ID NO:1 as described in the previous figures. Mean totalconcentration of lysoGb3 (m/z 786) and its analogs (m/z 784, m/z 802,and m/z 804) measured in plasma from a total of n=15 per group of eitherC57BL/6 (WT) or GLA null (KO) animals treated with vehicle, or GLA nullanimals treated with the indicated doses of rAAV carrying codonoptimized GLA of SEQ ID NO:1. Error bars indicate standard deviation. *indicates P<0.01 compared with vehicle-treated GLA null.

FIG. 12 . Gb3 Analysis in Tissues. Tissues were collected from n=6(hearts) or n=12 (kidney, small intestine, liver) from C57BL/6 (WT) orGLA-null (KO) mice treated with vehicle or increasing doses of rAAVcomprising (i) a capsid with a capsid protein of SEQ ID NO:4 and (ii) anucleic acid comprising a nucleotide sequence of SEQ ID NO:1 (asdescribed in the previous Figures) at Day 6 post-IV injection. Barsrepresent the mean response ratio of all Gb3 species measured. Errorbars represent standard deviation. * indicates P<0.005 when compared toGroup 2 (“KO-Vehicle”).

FIG. 13 graphically describes GLA activity in plasma of wild typeC57BL/6 mice following a single intravenous administration of 4D-310(rAAV comprising (i) a capsid with a capsid protein of SEQ ID NO:4 and(ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO:6) ateach of the specified doses.

FIG. 14 graphically describes GLA activity in specified tissues of wildtype C57BL/6 mice following a single intravenous administration of4D-310 (rAAV comprising (i) a capsid with a capsid protein of SEQ IDNO:4 and (ii) a nucleic acid comprising a nucleotide sequence of SEQ IDNO:6) at each of the specified doses.

FIG. 15 illustrates biodistribution of 4D-310 (by qPCR) to key Fabrytissues following a single intravenous administration of the specifieddose of 4D-310, vehicle control or C102.EGFP (rAAV comprising a capsidcomprising a capsid protein of SEQ ID NO:4 and a nucleic acid encodingEGFP) to non-human primates.

FIG. 16 illustrates AGA in NHP plasma, arranged by dosage, after asingle intravenous administration of 4D-310 at each of the specifieddoses.

FIG. 17 illustrates AGA in NHP plasma, arranged by animal, after asingle intravenous administration of 4D-310 at each of the specifieddoses

FIG. 18 illustrates fold-change in AGA (relative to vehicle control) inFabry-relevant NHP tissues, arranged by dosage, after a singleintravenous administration of 4D-310 at each of the specified doses

FIG. 19 illustrates fold-change in AGA (relative to vehicle control) inFabry-relevant NHP tissues, arranged by animal, after a singleintravenous administration of 4D-310 at the 5×10¹³ vg/kg dose.

FIG. 20 illustrates biodistribution of GLA RNA expression (by RT-qPCR)following a single intravenous administration of the specified dose of4D-310 to non-human primates.

FIGS. 21A-B IHC detection of AGA in heart tissue of vehicle-treated or4D-310-treated NHPs after a single IV administration at the 5×10¹³ vg/kgdose FIG. 21A shows AGA activity in the left ventricle; FIG. 21B showsAGA activity in the ventricular septum.

FIGS. 22A-B IHC detection of AGA in liver (FIG. 22A) and kidney (FIG.22B) tissues of vehicle-treated or 4D-310-treated NHPs after a single IVadministration at the 5×10¹³ vg/kg dose. Upregulation of AGA activity intreated animals over a high endogenous level in the vehicle animal isshown.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

A “codon adaptation index,” as used herein, refers to a measure of codonusage bias. A codon adaptation index (CAI) measures the deviation of agiven protein coding gene sequence with respect to a reference set ofgenes (Sharp P M and Li W H, Nucleic Acids Res. 15(3):1281-95 (1987)).CAI is calculated by determining the geometric mean of the weightassociated to each codon over the length of the gene sequence (measuredin codons):

$\begin{matrix}{{CAI} = {{\exp( {1/L{\sum\limits_{i = 1}^{L}{\ln( {w_{1}(l)} )}}} )}.}} & (I)\end{matrix}$

For each amino acid, the weight of each of its codons, in CAI, iscomputed as the ratio between the observed frequency of the codon (fi)and the frequency of the synonymous codon (fj) for that amino acid:

$\begin{matrix}{w_{i} = {{\frac{f_{i}}{\max( f_{j} )}{ij}} \in \lbrack {{synonymous}{codons}{for}{amino}{acid}} \rbrack}} & ({II})\end{matrix}$

The term “isolated” designates a biological material (cell, nucleic acidor protein) that has been removed from its original environment (theenvironment in which it is naturally present). For example, apolynucleotide present in the natural state in a plant or an animal isnot isolated, however the same polynucleotide separated from theadjacent nucleic acids in which it is naturally present, is considered“isolated.”

As used herein, a “coding region” or “coding sequence” is a portion ofpolynucleotide which consists of codons translatable into amino acids.Although a “% top codon” (TAG, TGA, or TAA) is typically not translatedinto an amino acid, it can be considered to be part of a coding region,but any flanking sequences, for example promoters, ribosome bindingsites, transcriptional terminators, introns, and the like, are not partof a coding region. The boundaries of a coding region are typicallydetermined by a stat codon at the 5′ terminus, encoding the aminoterminus of the resultant polypeptide, and a translation stop codon atthe 3′ terminus, encoding the carboxyl terminus of the resultingpolypeptide. Two or more coding regions can be present in a singlepolynucleotide construct, e.g., on a single vector, or in separatepolynucleotide constructs e.g., on separate (different) vectors. Itfollows, then that a single vector can contain just a single codingregion, or comprise two or more coding regions.

As used herein, the term “regulatory region” refers to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding region, and whichinfluence the transcription, RNA processing, stability, or translationof the associated coding region. Regulatory regions can includepromoters, translation leader sequences, introns, polyadenylationrecognition sequences, RNA processing sites, effector binding sites andstem-loop structures. If a coding region is intended for expression in aeukaryotic cell, a polyadenylation signal and transcription terminationsequence will usually be located 3′ to the coding sequence.

As used herein, the term “nucleic acid” is interchangeable with“polynucleotide” or “nucleic acid molecule” and a polymer of nucleotidesis intended.

A polynucleotide which encodes a gene product, e g., a polypeptide, caninclude a promoter and/or other transcription or translation controlelements operably associated with one or more coding regions. In anoperable association a coding region for a gene product, e.g., apolypeptide, is associated with one or more regulatory regions in such away as to place expression of the gene product under the influence orcontrol of the regulatory region(s). For example, a coding region and apromoter are “operably associated” if induction of promoter functionresults in the transcription of mRNA encoding the gene product encodedby the coding region, and if the nature of the linkage between thepromoter and the coding region does not interfere with the ability ofthe promoter to direct the expression of the gene product or interferewith the ability of the DNA template to be transcribed. Othertranscription control elements, besides a promoter, for exampleenhancers, operators, repressors, and transcription termination signals,can also be operably associated with a coding region to direct geneproduct expression.

“Transcriptional control sequences” refer to DNA regulatory sequences,such as promoters, enhancers, terminators, and the like, that providefor the expression of a coding sequence in a host cell. A variety oftranscription control regions are known to those skilled in the art.These include, without limitation, transcription control regions whichfunction in vertebrate cells, such as, but not limited to, promoter andenhancer segments from cytomegaloviruses (the immediate early promoter,in conjunction with intron-A), simian virus 40 (the early promoter), andretroviruses (such as Rous sarcoma virus). Other transcription controlregions include those derived from vertebrate genes such as actin, heatshock protein, bovine growth hormone and rabbit beta-globin, as well asother sequences capable of controlling gene expression in eukaryoticcells. Additional suitable transcription control regions includetissue-specific promoters and enhancers as well as lymphokine-induciblepromoters (e.g., promoters inducible by interferons or interleukins).

Similarly, a variety of translation control elements are known to thoseof ordinary skill in the art. These include, but are not limited toribosome binding sites, translation initiation and termination codons,and elements derived from picornaviruses (particularly an internalribosome entry site, or IRES, also referred to as a CITE sequence).

The term “expression” as used herein refers to a process by which apolynucleotide produces a gene product, for example, an RNA or apolypeptide. It includes without limitation transcription of thepolynucleotide into messenger RNA (mRNA), transfer RNA (tRNA), smallhairpin RNA (shRNA), small interfering RNA (siRNA) or any other RNAproduct, and the translation of an mRNA into a polypeptide. Expressionproduces a “gene product.” As used herein, a gene product can be eithera nucleic acid, e.g., a messenger RNA produced by transcription of agene, or a polypeptide which is translated from a transcript. Geneproducts described herein further include nucleic acids with posttranscriptional modifications, e.g., polyadenylation or splicing, orpolypeptides with post translational modifications, e.g., methylation,glycosylation, the addition of lipids, association with other proteinsubunits, or proteolytic cleavage.

A “vector” refers to any vehicle for the cloning of and/or transfer of anucleic acid into a host cell A vector can be a replicon to whichanother nucleic acid segment can be attached so as to bring about thereplication of the attached segment. The term “vector” includes bothviral and nonviral vehicles for introducing the nucleic acid into a cellin vitro, ex vivo or in vivo. A large number of vectors are known andused in the an including, for example, plasmids, modified eukaryoticviruses, or modified bacterial viruses. Insertion, of a polynucleotideinto a suitable vector can be accomplished by ligating the appropriatepolynucleotide fragments into a chosen vector that has complementarycohesive termini.

Vectors can be engineered to encode selectable markers or reporters thatprovide for the selection or identification of cells that haveincorporated the vector. Expression of selectable markers or reportersallows identification and/or selection of host cells that incorporateand express other coding regions contained on the vector. Examples ofselectable marker genes known and used in the art include: genesproviding resistance to ampicillin, streptomycin, gentamycin, kanamycin,hygromycin, bialaphos herbicide, sulfonamide, and the like; and genesthat are used as phenotypic markers, i.e., anthocyanin regulatory genes,isopentanyl tansferase gene, and the like. Examples of reporters knownand used in the art include: luciferase (Luc), green fluorescent protein(GFP), chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ),β-glucuronidase (Gus), and the like. Selectable markers can also beconsidered to be reporters.

Eukaryotic viral vectors that can be used include, but are not limitedto, adenovirus vectors, retrovirus vectors, adeno-associated virusvectors, poxvirus, e.g., vaccinia virus vectors, baculovirus vectors, orherpesvirus vectors. Non-viral vectors include plasmids, liposomes,electrically charged lipids (cytofectins), DNA-protein complexes, andbiopolymers.

“Promoter” and “promoter sequence” are used interchangeably and refer toa DNA sequence capable of controlling the expression of a codingsequence or functional RNA. In general, a coding sequence is located 3′to a promoter sequence. Promoters can be derived in their entirety froma native gene, or be composed of different elements derived fromdifferent promoters found in nature, or even comprise synthetic DNAsegments. It is understood by those skilled in the art that differentpromoters can direct the expression of a gene in different tissues orcell types, or at different stages of development, or in response todifferent environmental or physiological condition. Promoters that causea gene to be expressed in most cell types at most times are commonlyreferred to as “constitutive promoters.” Promoters that cause a gene tobe expressed in a specific cell type are commonly referred to as“cell-specific promoters” or “tissue-specific promoters.” Promoters thatcause a gene to be expressed at a specific stage of development or celldifferentiation are commonly referred to as “developmentally-specificpromoters” or “cell differentiation-specific promoters.” Promoters thatare induced and cause a gene to be expressed following exposure ortreatment of the cell with an agent, biological molecule, chemical,ligand, light, or the like that induces the promoter are commonlyreferred to as “inducible promoters” or “regulatable promoters.” It isfurther recognized that since in most cases the exact boundaries ofregulatory sequences have not been completely defined. DNA fragments ofdifferent lengths can have identical promoter activity.

The term “plasmid” refers to an extra-chromosomal element often carryinga gene that is not part of the central metabolism of the cell, andusually in the form of circular double-stranded DNA molecules. Suchelements can be autonomously replicating sequences, genome integratingsequences, phage or nucleotide sequences, linear, circular, orsupercoiled, of a single- or double-stranded DNA or RNA, derived fromany source, in which a number of nucleotide sequences have been joinedor recombined into a unique construction which is capable of introducinga promoter fragment and DNA sequence for a selected gene product alongwith appropriate 3′ untranslated sequence into a cell.

A polynucleotide or polypeptide has a certain percent “sequenceidentity” to another polynucleotide or polypeptide, meaning that, whenaligned, that percentage of bases or amino acids are the same whencomparing the two sequences. Sequence similarity can be determined in anumber of different manners. To determine sequence identity, sequencescan be aligned using the methods and computer programs, including BLAST,available over the world wide web at ncbi.nlm.nih.gov/BLAST/. Anotheralignment algorithm is FASTA, available in the Genetics Computing Group(GCG) package, from Madison, Wis., USA. Other techniques for alignmentare described in Methods in Enzymology, vol. 266: Computer Methods forMacromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press,Inc. Of particular interest are alignment programs that permit gaps inthe sequence. The Smith-Waterman is one type of algorithm that permitsgaps in sequence alignments. See Meth. Mol. Biol. 70: 173-187 (1997).Also, the GAP program using the Needleman and Wunsch alignment methodcan be utilized to align sequences. See J. Mol. Biol. 48: 443-453(1970).

The term “4D-310” refers to a recombinant AAV virion comprising (i) acapsid comprising a capsid protein with the amino acid of SEQ ID NO:4and (ii) a heterologous nucleic acid comprising the nucleotide sequenceset forth as SEQ ID NO:6. The nucleotide sequence of SEQ ID NO:6comprises the nucleotide sequence of SEQ ID NO:1 operably linked to aCAG promoter comprising the nucleotide sequence of SEQ ID NO.5.

The term “4D-C102” or “C102” refers to a variant AAV capsid proteincomprising the amino acid sequence of SEQ ID NO:4.

The terms “GLA” and “AGA” are used herein interchangeably to refer tothe gene encoding alpha-galactosidase and the encoded protein.

In one embodiment, the present invention provides a modified nucleicacid molecule comprising a nucleotide sequence that encodes apolypeptide of SEQ ID NO:2 (human AGA, consisting of 429 amino acids andavailable in GenBank Accession Nos X14448.1 and U78027), wherein thenucleic acid sequence has been codon optimized. In another embodiment,the starting nucleic acid sequence that encodes a polypeptide of SEQ IDNO:2 and that is subject to codon optimization has the nucleotidesequence set forth as SEQ ID NO:3. In preferred embodiments, thesequence that encodes a polypeptide of SEQ ID NO:2 is codon optimizedfor human expression. SEQ ID NO:1 is a codon optimized version of SEQ IDNO:3, optimized for human expression:

(SEQ ID NO: 1) ATGCAGCTGCGGAATCCTGAACTGCACCTGGGATGTGCCCTGGCTCTGAGATTTCTGGCCCTGGTGTCTTGGGACATCCCTGGCGCTAGAGCCCTGGATAATGGCCTGGCCAGAACACCTACAATGGGCTGGCTGCACTGGGAGAGATTCATGTGCAACCTGGACTGCCAAGAGGAACCCGACAGCTGCATCAGCGAGAAGCTGTTCATGGAAATGGCCGAGCTGATGGTGTCCGAAGGCTGGAAGGATGCCGGCTACGAGTACCTGTGCATCGACGACTGTTGGATGGCCCCTCAGAGAGACTCTGAGGGCAGACTGCAAGCCGATGCTCAGAGATTCCCTCACGGCATCAGACAGCTGGCCAACTACGTGCACAGCAAGGGCCTGAAGCTGGGCATCTATGCCGACGTGGGCAACAAGACCTGTGCCGGCTTTCCTGGCAGCTTCGGCTACTACGATATCGACGCCCAGACCTTCGCCGATTGGGGAGTCGATCTGCTGAAGTTCGACGGCTGCTACTGCGACAGCCTGGAAAATCTGGCCGACGGCTACAAGCACATGTCACTGGCCCTGAATCGGACCGGCACATCCATCGTGTACAGCTGCGAGTGGCCCCTGTACATGTGGCCCTTCCAGAAGCCTAACTACACCGAGATCAGACAGTACTGCAACCACTGGCGGAACTTGGCCGACATCGACGATAGCTGGAAGTCCATCAAGAGCATCCTGGACTGGACCAGCTTCAATCAAGAGCGGATCGTGGACGTGGCAGGACCTGGGGGATGGAACGATCCTGACATGCTGGTCATCGGCAACTTCGGCCTGAGCTGGAACCAGCAAGTGACCCAGATGGCCCTGTGGGCCATTATGGCCGCTCCTCTGTTCATGAGCAACGACCTGAGACACATCAGCCCTCAGGCCAAGGCTCTGCTCCAGGACAAGGATGTGATCGCTATCAACCAGGATCCTCTGGGCAAGCAGGGCTACCAGCTGAGACAGGGCGACAATTTCGAAGTGTGGGAAAGACCCCTGAGGGGACTGGCTTGGGCCGTGGCCATGATCAACAGACAAGAGATGGGCGGACCCCGGTCCTACACAATTGCCGTGGCTTCTCTCGGCAAAGGCGTGGCCTGTAATCCCGCCTGCTTTATCACACAGCTGCTGCCCGTGAAGAGAAAGCTGGGCTTTTACGAGTGGACCAGCAGACTGCGGAGCCACATCAATCCTACCGGCACAGTGCTGCTGCAACTGGAAAACACAATGCAGATGAGCCTGAAGGACCTGCTCTAA

The nucleotide sequence of SEQ ID NO:1 comprises a TAA stop codon. Inalternative embodiments, a nucleotide sequence comprising the first 1287nucleotides of SEQ ID NO:1 and ending with a different stop codon (e.g.TAG or TGA).

In one aspect, the disclosure provides a polynucleotide comprising thenucleotide sequence of SEQ ID NO:1 or polynucleotide comprising anucleotide sequence at least 90%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identical to the nucleotide sequenceof SEQ ID NO:1 and which encodes a human AGA polypeptide having theamino acid sequence of SEQ ID NO-2:

(SEQ ID NO: 2) MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKQLKLGIYADYGNKTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRNFADIDDSWKSIKSILDWTSFNQERIVDVAGPGQWNDPDMLVIGNFGLSWNQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDNFEVWERPLSGLAWAVAMINRQBIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSHINPTGTVLLQLENTMQMSLKDLL

The term “codon-optimized” as it refers to genes or coding regions ofnucleic acid molecules for transformation of various hosts, refers tothe alteration of codons in the gene or coding regions of the nucleicacid molecules to reflect the typical codon usage of the host organismwithout altering the polypeptide encoded by the DNA. Such optimizationincludes replacing at least one, or more than one, or a significantnumber, of codons with one or more codons that are more frequently usedin the genes of that organism.

Deviations in the nucleotide sequence that comprises the codons encodingthe amino acids of, any polypeptide chain allow for variations in thesequence coding for the gene. Since each codon consists of threenucleotides, and the nucleotides comprising DNA are restricted to fourspecific bases, there are 64 possible combinations of nucleotides, 61 ofwhich encode amino acids (the remaining three codons encode signalsending translation). The “genetic code” which shows which codons encodewhich amino acids is reproduced herein as Table 1. As a result, manyamino acids are designated by more than one codon. For example, theamino acids alanine and proline are coded for by four triplets, serineand arginine by six, whereas tryptophan and methionine are coded by justone triplet. This degeneracy allows for DNA base composition to varyover a wide range without altering the amino acid sequence of theproteins encoded by the DNA.

TABLE 1 TABLE-US-00001 The Standard Genetic CodeT C A G T TTT Phe (F) TCT Ser (S) TAT Tyr (Y) TGTCys (C) TTC Phe (F) TCC Ser (S) TAC Tyr (Y) TGCTTA Leu (L) TCA Ser (S) TAA Stop TGA Stop TTG Leu(L) TCG Ser (S) TAG Stop TGG Trp (W) C CTT Lea (L)CCT Pro (P) CAT His (H) CGT Arg (R) CTC Leu (L)CCC Pro (P) CAC His (H) CGC Arg (R) CTA Leu (L)CCA Pro (P) CAA Gln (Q) CGA Arg (R) CTG Les (L)CCG Pro (P) CAC Gln (Q) CGG Arg (R) A ATT Ile (I)ACT Thr (T) AAT Asn (N) AGT Ser (S) ATC Ile (I)ACC Thr (T) AAC Asn (N) AGC Ser (S) ATA Ile (I)ACA Thr (T) AAA Lys (K) AGA Arg (R) ATG Met (M)ACG Thr (T) AAG Lys (K) AGG Arg (R) G GTT Val (V)GCT Ala (A) GAT Asp (D) GGT Gly (G) GTC Val (V)GCC Ala (A) GAC Asp (D) GGC Gly (G) GTA Val (V)GCA Ala (A) GAA Glu (E) GGA Gly (G) GTG Val (V)GCG Ala (A) GAG Glu (E) GGG Gly (G)

Many organisms display a bias for use of particular codons to code forinsertion of a particular amino acid in a growing peptide chain. Codonpreference, or codon bias, differences in codon usage between organisms,is afforded by degeneracy of the genetic code, and is well documentedamong many organisms. Codon bias often correlates with the efficiency oftranslation of messenger RNA (mRNA), which is in turn believed to bedependent on, inter alia, the properties of the codons being translatedand the availability of particular transfer RNA (tRNA) molecules. Thepredominance of selected tRNAs in a cell is generally a reflection ofthe codons used most frequently in peptide synthesis. Accordingly, genescan be tailored for optimal gene expression in a given organism based oncodon optimization.

Given the large number of gene sequences available for a wide variety ofanimal, plant and microbial species, the relative frequencies of codonusage have been calculated. Codon usage tables are available, forexample, at the “Codon Usage Database” available atwww.kazusa.or.jp/codon/(visited Jun. 18, 2012). See Nakamura, Y., et al.Nucl. Acids Res. 28:292 (2000).

Randomly assigning codons at an optimized frequency to encode a givenpolypeptide sequence can be done manually by calculating codonfrequencies for each amino acid, and then assigning the codons to thepolypeptide sequence randomly. Additionally, various algorithms andcomputer software programs can be used to calculate an optimal sequence.

Non-Viral Vectors

In some embodiments, a non-viral vector (e.g. an expression plasmid)comprising a modified nucleic acid as herein described is provided.Preferably, the non-viral vector is a plasmid comprising a nucleic acidsequence of SEQ ID NO: 1, or a sequence at least 90% identical thereto.

Viral Vectors

In preferred embodiments, a viral vector comprising a modified (codonoptimized) nucleic acid as herein described is provided. Preferably, theviral vector comprises a nucleic acid sequence of SEQ ID NO: 1, or asequence at least 90% identical thereto, operably linked to anexpression control sequence. Examples of suitable viral vectors includebut are not limited to adenoviral, retroviral, lentiviral, herpesvirusand adeno-associated virus (AAV) vectors.

In a preferred embodiment, the viral vector includes a portion of aparvovirus genome, such as an AAV genome with the rep and cap genesdeleted and/or replaced by the modified GLA gene sequence and itsassociated expression control sequences. The modified human GLA genesequence is typically inserted adjacent to one or two (i.e., is flankedby) AAV TRs or TR elements adequate for viral replication (Xiao et al.,1997, J. Virol. 71(2): 941-948), in place of the nucleic acid encodingviral rep and cap proteins Other regulatory sequences suitable for usein facilitating tissue-specific expression of the modified GLA genesequence in the target cell may also be included.

Those skilled in the art will appreciate that an AAV vector comprising atransgene and lacking virus proteins needed for viral replication (e.g.,cap and rep), cannot replicate since such proteins are necessary forvirus replication and packaging. Helper viruses include, typically,adenovirus or herpes simplex virus. Alternatively, as discussed below,the helper functions (E1a, E1b, E2a, E4, and VA RNA) can be provided toa packaging cell including by transfecting the cell with one or morenucleic acids encoding the various helper elements and/or the cell cancomprise the nucleic acid encoding the helper protein. For instance, HEK293 were generated by transforming human cells with adenovirus 5 DNA andnow express a number of adenoviral genes, including, but not limited toE1 and E3 (see, e.g., Graham et al., 1977, J. Gen. Virol. 36:59-72).Thus, those helper functions can be provided by the HEK 293 packagingcell without the need of supplying them to the cell by, e.g., a plasmidencoding them.

The viral vector may be any suitable nucleic acid construct, such as aDNA or RNA construct and may be single stranded, double stranded, orduplexed (i.e., self complementary as described in WO 2001/92551).

The viral capsid component of the packaged viral vectors may be aparvovirus capsid. AAV Cap and chimeric capsids are preferred. Forexample, the viral capsid may be an AAV capsid (e.g., AAV1, AAV2, AAV3,AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11. AAV12, AAV1.1, AAV2.5,AAV6.1, AAV6.3.1, AAV9.45, AAVrh10, AAVrh74, RHM4-1, AAV2-TT,AAV2-TT-S312N, AAV3B-S312N, AAV-LK03, snake AAV, avian AAV, bovine AAV,canine AAV, equine AAV, ovine AAV, goat AAV, shrimp AAV, and any otherAAV now known or later discovered. see, e.g., Fields et al., VIROLOGY,volume 2, chapter 69 (4.sup.th.ed., Lippincott-Raven Publishers).

In some embodiments, the viral capsid component of the packaged viralvector is a variant of a native AAV capsid (i.e. comprises one or moremodifications relative to a native AAV capsid). In some embodiments, thecapsid is a variant of an AAV2, AAV5 or AAV8 capsid. In preferredembodiments, the capsid is a variant of an AAV2 capsid, such as thosedescribed in PCT application Ser. No. 18/51812 (WIPO Publication NumberWO 2019/060454 (e.g. comprising the amino acid sequence of any of SEQ IDNOs: 43-61), the contents of which are incorporated herein by reference.In a particularly preferred embodiment, the capsid comprises a capsidprotein having the following amino acid sequence:

(SEQ ID NO: 4) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNQLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSQTQKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSQLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKBVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTINNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSBYSWTGATKYHLNQRDSLVNPGPAMASHKDDEEKFFPQSQVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYQSVSTNLQRGNLANKTTNKDARQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSINVDFTVDINGVYSEPRPIGTRYLTRNL

A full complement of AAV Cap proteins includes VP1, VP2, and VP3. TheORF comprising nucleotide sequences encoding AAV VP capsid proteins maycomprise less than a full complement AAV Cap proteins or the fullcomplement of AAV Cap proteins may be provided.

In yet another embodiment the present invention provides for the use ofancestral AAV vectors for use in therapeutic in vivo gene therapy.Specifically, in silico-derived sequences were synthesized de novo andcharacterized for biological activities. This effort led to thegeneration of nine functional putative ancestral AAVs and theidentification of Anc80, the predicted ancestor of AAV serotypes 1, 2, 8and 9 (Zinn et al., 2015, Cell Reports 12:1056-1068). Predicting andsynthesis of such ancestral sequences in addition to assembling into avirus particle may be accomplished by using the methods described in WO2015/054653, the contents of which are incorporated by reference herein.Notably, the use of the virus particles assembled from ancestral viralsequences may exhibit reduced susceptibility to pre-existing immunity incurrent day human population than do contemporary viruses or portionsthereof.

The invention includes packaging cells, which are encompassed by “hostcells,” which may be cultured to produce packaged viral vectors of theinvention. The packaging cells of the invention generally include cellswith heterologous (1) viral vector function(s), (2) packagingfunction(s), and (3) helper function(s). Each of these componentfunctions is discussed in the ensuing sections.

Initially, the vectors can be made by several methods known to skilledartisans (see, e.g., WO 2013/063379). A preferred method is described inGrieger, et al. 2015, Molecular Therapy 24(2)-287-297, the contents ofwhich are incorporated by reference herein for all purposes. Briefly,efficient transfection of HEK293 cells is used as a starting point,wherein an adherent HEK293 cell line from a qualified clinical mastercell bank is used to grow in animal component-free suspension conditionsin shaker flasks and WAVE bioreactors that allow for rapid and scalablerAAV production. Using the triple transfection method (e.g., WO96/40240), the suspension HEK293 cell line generates greater than 10⁵vector genome containing particles (vg)/cell or greater than 10¹⁴ vg/Lof cell culture when harvested 48 hours post-transfection. Morespecifically, triple transfection refers to the fact that the packagingcell is transfected with three plasmids: one plasmid encodes the AAV repand cap genes, another plasmid encodes various helper functions (e.g.,adenovirus or HSV proteins such as E1a, E1b, E2a, E4, and VA RNA, andanother plasmid encodes the transgene and its various control elements(e.g., modified GLA gene and CAG promoter).

To achieve the desired yields, a number of variables are optimized suchas selection of a compatible serum-free suspension media that supportsboth growth and transfection, selection of a transfection reagent,transfection conditions and cell density. A universal purificationstrategy, based on ion exchange chromatography methods, was alsodeveloped that resulted in high purity vector preps of AAV serotypes1-6, 8, 9 and various chimeric capsids. This user-friendly process canbe completed within one week, results in high full to empty particleratios (>90% full particles), provides post-purification yields(>1×10{circumflex over ( )}13 vg/L) and purity suitable for clinicalapplications and is universal with respect to all serotypes and chimericparticles. This scalable manufacturing technology has been utilized tomanufacture GMP Phase I clinical AAV vectors for retinalneovascularization (AAV2), Hemophilia B (scAAV8), Giant AxonalNeuropathy (scAAV9) and Retinitis Pigmentosa (AAV2), which have beenadministered into patients. In addition, a minimum of a 5-fold increasein overall vector production by implementing a perfusion method thatentails harvesting rAAV from the culture media at numerous time-pointspost-transfection.

The packaging cells include viral vector functions, along with packagingand vector functions. The viral vector functions typically include aportion of a parvovirus genome, such as an AAV genome, with rep and capdeleted and replaced by the modified GLA sequence and its associatedexpression control sequences. The viral vector functions includesufficient expression control sequences to result in replication of theviral vector for packaging. Typically, the viral vector includes aportion of a parvovirus genome, such as an AAV genome with rep and capdeleted and replaced by the transgene and its associated expressioncontrol sequences. The transgene is typically flanked by two AAV TRs, inplace of the deleted viral rep and cap ORFs. Appropriate expressioncontrol sequences are included, such as a tissue-specific promoter andother regulatory sequences suitable for use in facilitatingtissue-specific expression of the transgene in the target cell. Thetransgene is typically a nucleic acid sequence that can be expressed toproduce a therapeutic polypeptide or a marker polypeptide.

The terminal repeats (TR(s))(resolvable and non-resolvable) selected foruse in the viral vectors am preferably AAV sequences, with serotypes 1,2, 3, 4, 5 and 6 being preferred. Resolvable AAV TRs need not have awild-type TR sequence (e.g., a wild-type sequence may be altered byinsertion, deletion, truncation or missense mutations), as long as theTR mediates the desired functions, e.g., vims packaging, integration,and/or provirus rescue, and the like. The TRs may be synthetic sequencesthat function as AAV inverted terminal repeats, such as the “double-Dsequence” as described in U.S. Pat. No. 5,478,745 to Samulski et al.,the entire disclosure of which is incorporated in its entirety herein byreference. Typically, but not necessarily, the TRs are from the sameparvovirus, e.g., both TR sequences are from AAV2.

The packaging functions include capsid components. The capsid componentsare preferably from a parvoviral capsid, such as an AAV capsid or achimeric AAV capsid function. Examples of suitable parvovirus viralcapsid components are capsid components from the family Parvoviridae,such as an autonomous parvovirus or a Dependovirus. For example, thecapsid components may be selected from AAV capsids, e.g., AAV1, AAV2,AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh10,AAVrh74, RHM4-1, RHM15-1, RHM15-2, RHM15-3/RHM15-5, RHM15-4, RHM15-6,AAV Hu.26, AAV1.1, AAV2.5, AAV6.1, AAV6.3.1, AAV9.45, AAV2i8, AAV2G9,AAV2i8G9, AAV2-TT, AAV2-TT-S312N, AAV3B-S312N, and AAV-LK03, and othernovel capsids as yet unidentified or from non-human primate sources.Capsid components may include components from two or more AAV capsids.

The packaged viral vector generally includes the modified GLA genesequence and expression control sequences flanked by TR elements,referred to herein as the “transgene” or “transgene expressioncassette,” sufficient to result in packaging of the vector DNA andsubsequent expression of the modified GLA gene sequence in thetransduced cell. The viral vector functions may, for example, besupplied to the cell as a component of a plasmid or an amplicon. Theviral vector functions may exist extrachromosomally within the ccli lineand/or may be integrated into the cell's chromosomal DNA.

Any method of introducing the nucleotide sequence carrying the viralvector functions into a cellular host for replication and packaging maybe employed, including but not limited to, electroporation, calciumphosphate precipitation, microinjection, cationic or anionic liposomes,and liposomes in combination with a nuclear localization signal. Inembodiments wherein the viral vector functions are provided bytransfection using a virus vector, standard methods for producing viralinfection may be used.

The packaging functions include genes for viral vector replication andpackaging. Thus, for example, the packaging functions may include, asneeded, functions necessary for viral gene expression, viral vectorreplication, rescue of the viral vector from the integrated state, viralgene expression, and packaging of the viral vector into a viralparticle. The packaging functions may be supplied together or separatelyto the packaging cell using a genetic construct such as a plasmid or anamplicon, a Baculovirus, or HSV helper construct. The packagingfunctions may exist extrachromosomally within the packaging cell, butare preferably integrated into the cell's chromosomal DNA. Examplesinclude genes encoding AAV Rep and Cap proteins.

The helper functions include helper virus elements needed forestablishing active infection of the packaging cell, which is requiredto initiate packaging of the viral vector. Examples include functionsderived from adenovirus, baculovirus and/or herpes virus sufficient toresult in packaging of the viral vector. For example, adenovirus helperfunctions will typically include adenovirus components E1a, E1b, E2a,E4, and VA RNA. The packaging functions may be supplied by infection ofthe packaging cell with the required virus. The packaging functions maybe supplied together or separately to the packaging cell using a geneticconstruct such as a plasmid or an amplicon. See, e.g., pXR helperplasmids as described in Rabinowitz et al., 2002, J. Virol. 76.791, andpDG plasmids described in Grimm el al., 1998, Human Gene Therapy9:2745-2760. The packaging functions may exist extrachromosomally withinthe packaging cell, but aw preferably integrated into the cell'schromosomal DNA (e.g., E1 or E3 in HEK 293 cells).

Any suitable helper virus functions may be employed. For example, wherethe packaging cells are insect cells, baculovirus may serve as a helpervirus. Herpes virus may also be used as a helper virus in AAV packagingmethods. Hybrid herpes viruses encoding the AAV Rep protein(s) mayadvantageously facilitate for mom scalable AAV vector productionschemes.

Any method of introducing the nucleotide sequence carrying the helperfunctions into a cellular host for replication and packaging may beemployed, including but not limited to, electroporation, calciumphosphate precipitation, microinjection, cationic or anionic liposomes,and liposomes in combination with a nuclear localization signal. Inembodiments wherein the helper functions are provided by transfectionusing a virus vector or infection using a helper virus; standard methodsfor producing viral infection may be used.

Any suitable permissive or packaging cell known in the art may beemployed in the production of the packaged viral vector. Mammalian cellsor insect cells are preferred. Examples of cells useful for theproduction of packaging cells in the practice of the invention include,for example, human cell lines, such as VERO, WI38, MRC5, A549, HEK 293cells (which express functional adenoviral E1 under the control of aconstitutive promoter), B-50 or any other HeLa cells, HepG2, Saos-2,HuH7, and HT1080 cell lines. In one aspect, the packaging cell iscapable of growing in suspension culture, more preferably, the cell iscapable of growing in serum-free culture. In one embodiment, thepackaging cell is a HEK293 that grows in suspension in serum freemedium. In another embodiment, the packaging cell is the HEK293 celldescribed in U.S. Pat. No. 9,441,206 and deposited as ATCC No. PTA13274. Numerous rAAV packaging cell lines are known in the at,including, but not limited to, those disclosed in WO 2002/46359. Inanother aspect, the packaging cell is cultured in the form of a cellstack (e.g. 10-layer cell stack seeded with HEK293 cells).

Cell lines for use as packaging cells include insect cell lines. Anyinsect cell which allows for replication of AAV and which can bemaintained in culture can be used in accordance with the presentinvention. Examples include Spodoptera frugiperda, such as the Sf9 orSf21 cell lines, Drosophila spp. cell lines, or mosquito cell lines,e.g., Aedes albopictus derived cell lines. A preferred cell line is theSpodoptera frugiperda Sf9 cell line. The following references areincorporated herein for their teachings concerning use of insect cellsfor expression of heterologous polypeptides, methods of introducingnucleic acids into such cells, and methods of maintaining such cells inculture: Method % in Molecular Biology, ed. Richard, Humana Press, N J(1995); O'Reilly et al., Baculovirus Expression Vectors: A LaboratoryManual, Oxford Univ. Press (1994); Samulski et al., 1989, J. Virol.63:3822-3828; Kajigaya et al., 1991, Proc. Natl. Acad. Sci. USA88-4646-4650; Ruffing et al., 1992, J. Virol. 66:6922-6930; Kimbauer etal., 1996, Virol. 219:37-44; Zhao et al., 2000, Virol. 272:382-393: andSamulski et al., U.S. Pat. No. 6,204,059.

Virus capsids according to the invention can be produced using anymethod known in the at, e.g., by expression from a baculovirus (Brown etal., (1994) Virology 198:477.488). As a further alternative, the virusvectors of the invention can be produced in insect cells usingbaculovirus vectors to deliver the rep/cap genes and rAAV template asdescribed, for example, by Urabe et al., 2002, Human Gene Therapy13:1935-1943.

In another aspect, the present invention provides for a method of rAAVproduction in insect cells wherein a baculovirus packaging system orvectors may be constructed to carry the AAV Rep and Cap coding region byengineering these genes into the polyhedrin coding region of abaculovirus vector and producing viral recombinants by transfection intoa host cell. Notably when using Baculovirus production for AAV,preferably the AAV DNA vector product is a self-complementary AAV likemolecule without using mutation to the AAV ITR. This appears to be aby-product of inefficient AAV rep nicking in insect cells which resultsin a self-complementary DNA molecule by virtue of lack of functional Repenzyme activity. The host cell is a baculovirus-infected cell or hasintroduced therein additional nucleic acid encoding baculovirus helperfunctions or includes these baculovirus helper functions therein. Thesebaculovirus viruses can express the AAV components and subsequentlyfacilitate the production of the capsids.

During production, the packaging cells generally include one or moreviral vector functions along with helper functions and packagingfunctions sufficient to result in replication and packaging of the viralvector. These various functions may be supplied together or separatelyto the packaging cell using a genetic construct such as a plasmid or anamplicon, and they may exist extrachromosomally within the cell line orintegrated into the cell's chromosomes.

The cells may be supplied with any one or more of the stated functionsalready incorporated, e.g., a cell line with one or more vectorfunctions incorporated extrachromosomally or integrated into the cell'schromosomal DNA, a cell line with one or more packaging functionsincorporated extrachromosomally or integrated into the cell'schromosomal DNA, or a cell line with helper functions incorporatedextrachromosomally or integrated into the cell's chromosomal DNA.

The rAAV vector may be purified by methods standard in the art such asby column chromatography or cesium chloride gradients. Methods forpurifying rAAV vectors are known in the art and include methodsdescribed in Clark et al., 1999, Human Gene Therapy 10(6):1031-1039;Schenpp and Clark, 2002, Methods Mol. Med. 69:427-443; U.S. Pat. No.6,566,118 and WO 98/09657.

Treatment Methods

In certain embodiments, a method is provided for the treatment of Fabrydisease n a subject in need of such treatment by administering to thesubject a therapeutically effective amount of a nucleic acid having anucleotide sequence at least 90%, at least 95%, at least 98% identical,or 100% identical to the nucleotide sequence of SEQ ID NO:1 or apharmaceutical composition comprising such a nucleic acid and at leastone pharmaceutically acceptable excipient. In some aspects, a nucleicacid having a nucleotide sequence at least 90% identical to SEQ ID NO:1is administered to a subject in an amount effective to reduce the levelof globotriaosylceramide (Gb3) in the subject.

In related aspects, a nucleic acid comprising a nucleotide sequence atleast 90%, at least 95%, at least 98% identical, or 100% identical tothe nucleotide sequence of SEQ ID NO:1 for use in the treatment of Fabrydisease is provided.

In other related aspects, the use of a nucleic acid comprising anucleotide sequence at least 90%, at least 95%, at least 98% identical,or 100% identical to the nucleotide sequence of SEQ ID NO:1 for themanufacture of a medicament is provided.

In other related aspects, the use of a nucleic acid comprising anucleotide sequence at least 90%, at least 95%, at least 98% identical,or 100% identical to the nucleotide sequence of SEQ ID NO:1 for themanufacture of a medicament for the treatment of Fabry disease isprovided.

In some aspects, the nucleotide sequence at least 90%, at least 95%, atleast 98% identical, or 100% identical to the nucleotide sequence of SEQID NO:1 is operably linked to an expression control sequence.

In preferred embodiments, a method for treating Fabry disease isprovided comprising administering to a subject in need thereof atherapeutically effective amount of a nucleic acid comprising thenucleotide sequence of SEQ ID NO:1 operably linked to a CAG promoter orby administering to the subject a pharmaceutical composition comprisingsuch a nucleic acid.

In other embodiments, a nucleic acid comprising the nucleotide sequenceof SEQ ID NO:1 operably linked to a CAG promoter for use in thetreatment of Fabry disease is provided. In some aspects, the CAGpromoter comprises a sequence that is at least 90%, at least 95%, or atleast 98% identical, or is 100% identical to the nucleotide sequence ofSEQ ID NO:5.

In other embodiments, the use of a nucleic acid comprising thenucleotide sequence of SEQ ID NO:1 operably linked to a CAG promoter forthe manufacture of a medicament for the treatment of Fabry disease isprovided.

In related aspects, a recombinant AAV (rAAV) virion comprising (i) anucleic acid having a nucleotide sequence at least 90%, at least 95%, atleast 98% identical, or 100% identical to the nucleotide sequence of SEQID NO:1 operably linked to an expression control sequence and (ii) anative or variant AAV capsid or a pharmaceutical composition comprisingsuch an rAAV for use in the treatment of Fabry disease, or for use inthe manufacture of a medicament for the treatment of Fabry disease, isprovided.

In some embodiments, the rAAV virion comprises a native AAV1, AAV2, AAV6or AAV8 capsid. In other embodiments, the rAAV virion comprises avariant AAV capsid that comprises one or mom modifications relative toAAV1, AAV2, AAV6, or AAV8. In a preferred embodiment, the AAV capsidcomprises the sequence of SEQ ID NO:4 or a sequence at least 95%, atleast 98% or at least 99% identical thereto.

In a preferred embodiment, the use of an rAAV in the treatment of Fabrydisease or for the manufacture of a medicament for the treatment ofFabry disease is provided, wherein the rAAV comprises (i) a nucleic acidcomprising a nucleotide sequence of SEQ ID NO:1 operably linked to a CAGpromoter and (ii) a capsid comprising a capsid protein having the aminoacid sequence of SEQ ID NO:4. In particularly preferred embodiments, therAAV comprises (i) a nucleic acid comprising a nucleotide sequence ofSEQ ID NO:6 and (ii) a capsid comprising a capsid protein having theamino acid sequence of SEQ ID NO:4. In some aspects, the rAAV isadministered by intramuscular and/or intravascular (e.g. intravenous)injection. In a particularly preferred embodiment, the rAAV isadministered as a single intravenous administration.

In other aspects, a pharmaceutical composition is provided comprising anucleic acid having a nucleotide sequence at least 90%, at least 95%, atleast 98% identical, or 100% identical to the nucleotide sequence of SEQID NO:1, optionally operably linked to an expression control sequence,and at least one pharmaceutically acceptable excipient. In someembodiments, the pharmaceutical composition comprises a nucleic acidcomprising the nucleotide sequence of SEQ ID NO:1 operably linked to aCAG promoter. In some embodiments, the CAG promoter comprises a sequenceat least 90%, at least 95%, or at least 98% identical to SEQ ID NO:5 oris identical to SEQ ID NO:5.

In other aspects, a pharmaceutical composition is provided comprising atleast one pharmaceutically acceptable excipient and an infectious rAAVcomprising (i) a nucleic acid comprising a nucleotide sequence of SEQ IDNO:1 operably linked to a CAG promoter and (ii) a capsid comprising acapsid protein having the amino acid sequence of SEQ ID NO.4. In relatedaspects, the infectious rAAV comprises (i) a capsid comprising a capsidprotein having the amino acid sequence of SEQ ID NO:4 and (ii) a nucleicacid comprising from 5′ to 3′: (a) an AAV2 terminal repeat (b) a CAGpromoter (c) a nucleic acid according to claim 3 (d) a polyadenylationsequence and (e) an AAV2 terminal repeat. In a particularly preferredembodiment, the infectious rAAV comprises (i) a capsid comprising acapsid protein having the amino acid sequence of SEQ ID NO:4 and (ii) anucleic acid comprising the sequence of SEQ ID NO:6 or a sequence atleast 90%, at least 95%, at least 98% or at least 99% identical thereto:

(SEQ ID NO: 6) TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTATCGATTGAATTCCCCGGGGATCCACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGGGCGCGCCAGGCGGGGGGGGGGGGGGCGAGGGGCGGGGGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGGGGGGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGGAGTCGCTGCGACGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGGGGGGGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGGGCCCTTTGTGGGGGGGGAGGGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCCCCGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGGGGTGCCCCGCGGTGCGGGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGTCGGTGGGGCTGCAACCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGGGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGTCTAGAGTCGACCTGCAGGTGGATATCTTGCTAGCACGCCACCATGCAGCTGCGGAATCCTGAACTGCACCTGGGATGTGCCCTGGCTCTGAGATTTCTGGCCCTGGTGTCTTGGGACATCCCTGGCGCTAGAGCCCTGGATAATGGCCTGGCCAGAACACCTACAATGGGCTGGCTGCACTGGGAGAGATTCATGTGCAACCTGGACTGCCAAGAGGAACCCGACAGCTGCATCAGCGAGAAGCTGTTCATGGAAATGGCCGAGCTGATGGTGTCCGAAGGCTGGAAGGATGCCGGCTACGAGTACCTGTGCATCGACGACTGTTGGATGGCCCCTCAGAGAGACTCTGAGGGCAGACTGCAAGCCGATCCTCAGAGATTCCCTCACGGCATCAGACAGCTGGCCAACTACGTGCACAGCAAGGGCCTGAAGCTGGGCATCTATGCCGACGTGGGCAACAAGACCTGTGCCGGCTTTCCTGGCAGCTTCGGCTACTACGATATGGACGCCCAGACCTTCGCCGATTGGGGAGTCGATCTGCTGAAGTTCGACGGCTGCTACTGCGACAGCCTGGAAAATCTGGCCGACGGCTACAAGCACATGTCACTGGCCCTGAATCGGACCGGCAGATCCATCGTGTACAGCTGCGAGTGGCCCCTGTACATGTGGCCCTTCCAGAAGCCTAACTACACCGAGATCAGACAGTACTGCAACCACTGGCGGAACTTCGCCGACATCGACGATAGCTGGAAGTCCATCAAGAGCATCCTGGACTGGACCAGCTTCAATCAAGAGCGGATCGTGGACGTGGCAGGACCTGGCGGATGGAACGATCCTGACATGCTGGTCATCGGCAACTTCGGCCTGAGCTGGAACCAGCAAGTGACCCAGATGGCCCTGTGGGCCATTATGGCCGCTCCTCTGTTCATGAGCAACGACCTGAGACACATCAGCCCICAGGCCAAGGCTCTGCTCCAGGACAAGGATGTGATCGCTATCAACCAGGATCCTCTGGGCAAGCAGGGCTACCAGCTGAGACAGGGCGACAATTTCGAAGTGTGGGAAAGACCCCTGAGCGGACTGGCTTGGGCCGTGGCCATGATCAACAGACAAGAGATCGGCGGACCCCGGTCCTACACAATTGCCGTGGCTTCTCTCGGCAAAGGCGTGGCCTGTAATCCCGCCTGCTTTATCACACAGCTGCTGCCCGTGAAGAGAAAGCTGGGCTTTTACGAGTGGACCAGCAGACTGCGGAGCCACATCAATCCTACCGGCACAGTGCTGCTGCAACTGGAAAACACAATGCAGATGAGCCTGAAGGACCTGCTCTAAGCCACGCGTAACACGTGCATGCGAGAGATCTGCGGCCGCGAGCTCGGGGATCCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTATGGCTGATTATGATCAATGCATCCTAGCCGGAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGGGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAG AGAGGGAGTGGCCAA

In some preferred embodiments, a human subject with Fabry disease isadministered a pharmaceutical composition comprising a pharmaceuticallyacceptable carrier and an rAAV vector comprising (i) a capsid comprisinga capsid protein comprising or consisting of the amino acid sequence setforth as SEQ ID NO:4 and (ii) a heterologous nucleic acid comprising thesequence of SEQ ID NO:6, wherein the subject is administered one or moredoses of the rAAV vector, each dose comprising from about 1×10¹² toabout 1×10¹⁵ vector particles/kg or vector genomes/kg, 1×10¹² to 1×10¹⁵vector particles or vector genomes, or about 1×10¹², about 2×10¹²,3×10¹², about 4×10¹², about 5×10¹², about 6×10¹², about 7×10¹², about8×10¹², about 9×10¹², about 1×10¹³, about 2×10¹³, about 3×10¹³, about4×10¹³, about 5×10¹³, about 6×10¹³, about 7×10¹³, about 8×10¹³, about9×10¹³, about 1×10¹⁴, about 2×10¹⁴, about 3×10¹⁴, about 4×10¹⁴, about5×10¹⁴, about 6×10¹⁴, about 7×10¹⁴, about 8×10¹⁴, about 9×10¹⁴ or about1×10¹⁵ vector particles/kg or vector genomes/kg. In some particularlypreferred aspects, the subject is administered one or more doses of therAAV, each dose comprising from about 1×10¹² vg/kg to 1×10¹⁴ vg/kg. e.g.about 3×10¹², about 1×10¹³, 3×10¹³, or about 5×10¹³ vector particles/kgor vector genomes/kg. In some particularly preferred embodiments, thepharmaceutical composition is administered to a human with Fabry diseasevia a single intravenous injection, wherein the single intravenousinjection is effective to treat Fabry disease in the human subject. Inother embodiments, the pharmaceutical composition is administered to ahuman with Fabry disease via a single intramuscular injection.

In some preferred embodiments, a pharmaceutical composition is provided,the pharmaceutical composition comprising a pharmaceutically acceptablecarrier and an rAAV vector comprising (i) a capsid comprising a capsidprotein comprising or consisting of the amino acid sequence set forth asSEQ ID NO:4 and (ii) a heterologous nucleic acid comprising the sequenceof SEQ ID NO:6, wherein the pharmaceutical composition comprises 1×10¹⁰to 1×10¹⁷ vector particles or vector genomes, 1×10¹³ to 1×10¹⁶ vectorparticles or vector genomes, or about 1×10¹³, about 2×10¹³, 3×10¹³,about 4×10¹³, about 5×10¹³, about 6×10¹³, about 7×10¹³, about 8×10¹³,about 9×10¹³, about 1×10¹⁴, about 2×10¹⁴, about 3×10¹⁴, about 4×10¹⁴,about 5×10¹⁴, about 6×10¹⁴, about 7×10¹⁴, about 8×10¹⁴, about 9×10¹⁴,about 1×10¹⁵, about 2×10¹⁵, about 3×10¹⁵, about 4×10¹⁵, about 5×10¹⁵,about 6×10¹⁵, about 7×10¹⁵, about 8×10¹⁵, about 9×10¹⁵, about 1×10¹⁶,about 5×10¹⁶ or about 8×10¹⁶ vector particles or vector genomes.

EXAMPLES

The following examples illustrate preferred embodiments of the presentinvention and are not intended to limit the scope of the invention inany way. While this invention has been described in relation to itspreferred embodiments, various modifications thereof will be apparent toone skilled in the art from reading this application.

Example 1—Codon Optimization of GLA cDNA Sequence

The human GLA open reading frame cDNA sequence, including the endogenousGLA secretion signal (GenBank Accession No. NM_000169.3; SEQ ID NO-3)was codon optimized for human expression. The optimization algorithmincluded parameters including, but not limited to, codon usage bias, GCcontent, CpG dinucleotides content, negative CpG islands, mRNA secondarystructure, RNA instability motifs, cryptic splicing sites, prematurepolyadenylation sites, internal chi sites and ribosomal binding sites,and repeat sequences.

The codon usage bias in humans was changed by upgrading the codonadaptation index (CAI) from 0.75 to 0.93. The average GC content wasoptimized from 48.6% in the native sequence to 57.9% in the optimizedsequence to prolong the half-life of the mRNA. Stem-Loop structures,which impact ribosomal binding and stability of mRNA, were broken in theoptimized sequence. In addition, negative cis-acting sites were screenedand deleted to optimize expression of the gene in human cells andseveral restriction enzyme sites were deleted.

The resulting codon optimized nucleotide sequence, set forth herein asSEQ ID NO:1, contains improved codon usage, altered GC content, bettermRNA stability, and modification of negative cis acting elements.

Example 2—Codon Optimized GLA cDNA Sequence is Expressed at HigherLevels in Cardiomyocytes from Patients with Fabry Disease

A human in vitro model system was generated to evaluate expression ofhuman GLA nucleic acid having the nucleotide sequence of SEQ ID NO.1 indiseased human cardiomyocytes derived from a human Fabry disease patientand functional correction of the disease phenotype.

Materials and Methods

Fibroblast Cell Culture and Reprogramming to Induced Pluripotent StemCell Lines

Non-diseased fibroblasts or male human Fabry disease fibroblasts wereobtained from Coriell Institute and cultured in Eagle's ModifiedEssential Medium (EMEM) with 15% Fetal Bovine Seam (FBS, Hyclone) and 1%Penicillin/Streptomycin (ThermoFisher). For reprogramming, cells werepassaged using 0.05% trypsin and plated at a density of 2.5×10⁴ cellsper cm² in 6 well tissue culture plates. Cells were kept at 37° C., 5%CO₂ in normoxic conditions.

Cellular reprogramming of diseased fibroblasts was performed by a singleRNA transfection of Oct4-Klf4-Sox2-Glis1 polycistronic transcriptaccording to the manufacturer's instructions (Simplicon RNAReprogramming Kit, EMD Millipore). At day 10, approximately 5×10⁴-1×10⁵reprogrammed cells were m-plated on growth factor reduced Matrigel(Corning) in mouse embryonic fibroblasts (MEF)-conditioned mediumcontaining B18R protein (200 ng/mL) supplemented with human iPSCReprogramming Boost Supplement II (EMD Millipore). At day 20,reprogrammed cells, recognized by altered morphology and ability to formsmall colonies, were transitioned to mTeSR-1 media (Stem CellTechnologies). Colonies of approximately 200 cells or larger wereisolated manually and plated on Matrigel coated plates in mTeSR-1medium. Fabry-iPSC lines were expanded from a single colony. TheFabry-iPSC lines were cultured on Matrigel in mTeSR-1 maintenance mediumand sub-cultured using Gentle Cell Dissociation Reagent (Stem CellTechnologies), every 4-5 days at 70-80% confluence. To ensure randomdifferentiation into all three germ layers, iPSC embryoid bodies (BBs)were formed in suspension culture for one week and then differentiatedin adherent conditions for an additional four weeks in 20% knockoutserum replacement in DMEM containing 1× GlutaMax, 1× non-essential aminoacids, with 1.4 μL/100 mL media of beta-mercaptoethanol (Thermo FisherScientific). iPSC clones used in cardiac differentiation were submittedto Cell Line Genetics for standard karyotyping, according to companyprotocols.

Fabry Diseased iPSC Cardiomyocyte Differentiation

Fabry iPSCs were seeded at 25,000 cells/cm² in mTeSR-1 in 3 twelve wellplates coated with growth factor reduced Matrigel (GFR Matrigel,Corning). Upon reaching the confluency, cultures were subjected tosequential GSK3β and Porcupine inhibition of the Wat pathway in RMPI1640 with B27 supplement without insulin (RB−, ThermoFisher). On day 6cells were fed with RMPI 1640 plus B27 supplement (RB+, Thermofisher).Fabry diseased cardiomyocytes that exhibited visible beating by day 15were passaged at 1:2 ratio into twelve well plates coated with GFRMatrigel in RB+. Following passage, Fabry cardiomyocytes were purifiedthrough glucose deprivation following a previously publisheddifferentiation paradigm (Lian et al., PNAS, 109(27):E1848-57). Initialbeating appeared 7 days after passage. After two weeks postpurification, Fabry diseased cardiomyocytes were used for cellcharacterization and transduction with recombinant AAV (rAAV) particlescomprising (i) a capsid with a capsid protein of SEQ ID NO:4 and (ii) anucleic acid comprising a nucleotide sequence of SEQ ID NO:1 operablylinked to a CAO promoter of SEQ ID NO:5.

Fabry Diseased iPSC Cardiomyocyte Transduction

Cardiomyocytes were transduced 14 days after passage into experimentalplates with the rAAV. On the day of transduction three wells wereharvested to obtain a cell count. Cells were transduced at Multiplicityof infections (MOI) of 25, 100, 250, based on the cell count and viraltiter. Vehicle was added in the highest volume. Cells were incubatedwith virus for 48 hours and then received a media change. The media waschanged every day other day until harvest, six days post transduction.

Immunocytochemistry (ICC)

iPSC and Germ Layer ICC

iPSCs or 30-day old plated EBs were washed once with Phosphate BufferedSaline without Magnesium or Calcium (PBS−/−) and fixed with 4%paraformaldehyde for 15 minutes at room temperature. Cells were thenwashed 3 times with PBS−/− and blocked for 30 minutes with 2% bovineserum albumin and 5% goat serum in 0.2% Triton X-100 in PBS−/−. Nanog,Oct-4, Sox-2 or β-tubulin 11, HNF-α and α-SMA primary antibodies wereincubated at room temperature for 2 hours followed by a goat anti-mouseor goat anti-rabbit AlexaFluor (ThermoFisher) secondary antibodies for 1hour at room temperature. Cells were counterstained with DAPI tovisualize the nuclei and were imaged on a Zeiss Axiovert. A1 invertedmicroscope.

Cardiomyocyte ICC

Six days post infection, cells were washed once with PBS−/− and fixedwith 4% paraformaldehyde for 15 minutes at room temperature Cells werethen washed 3 times with PBS−/− and blocked for 30 minutes with 2%bovine serum albumin and 5% goat serum in 0.2% Triton X-100 in PBS−/−.Primary antibodies against alpha galactosidase A (Abnova) and cardiactroponin T (cTNT, R&D) were incubated with cells in blocking solutionfor 2 hours at room temperature followed by a goat anti-mouseAlexaFluor-555 (ThermoFisher) secondary antibody for 1 hour at roomtemperature. Additionally, conjugated CD77 (Gb3)-AlexaFluor647 (BD) wasincubated with fixed cells in blocking buffer for one hour. Cells werewashed 3 times and counterstained with DAPI or Hoechst 33342 tovisualize the nuclei and were imaged on a Zeiss Axiovert.A1 invertedmicroscope.

Western Blot

Six days post infection, cells were lifted with 0.05% trypsin andpelleted at 300×g for 5 minutes. Lysates were made using RIPA bufferplus protease inhibitors. Lysates were incubated on ice for 15 minutesand centrifuged at 21,000×g for 15 minutes. Supernatants were collected,and a bicinchoninic acid assay (BCA) was run to determine proteinconcentration Concentrations were normalized and gels were equallyloaded. An SDS-PAGE was run using a 4-12% polyacrylamide gel at 200volts for 30 minutes. Protein was transferred to a 0.2 μm nitrocellulosemembrane and probed with anti-alpha galactosidase A (Atlas) anti-GAPDH(Stem Cell Technologies) antibodies followed by species specifichorseradish peroxidase secondary antibodies overnight. Enhancedchemilumenescence substrate was used to develop protein bands, and banddetection was captured on a BioRad ChemiDoc MP.

AGA Activity Assay

The AGA activity assay was performed with an alpha-galactosidasesynthetic fluorometric substrate (BioVision, K407). AGA activity assaywas performed according to the manufacturer's protocol with thefollowing changes. Cells were dissociated using 0.25% trypsin andcentrifuged at 300×g for 3 minutes. Cells were lysed withalpha-galactosidase buffer and incubated on ice for 10 minutes. Sampleswere centrifuged at 12,000×g for 10 minutes at 4° C. and supernatant wasstored at −80° C. The following day, samples were thawed on ice andprotein quantification via BCA assay was performed to allow for proteinconcentration normalization during analysis. The AGA activity assayreaction was incubated for 1 hour at room temperature and stopped usingappropriate volume stop buffer AGA activity assay reaction wasimmediately read on a Cytation3 plate reader (BioTek) at 360excitation/445 emission with a gain set to highest concentration of AGAstandard curve.

TABLE 2 List of Antibodies Antibody Host Company-Catalog No. DilationPrimary Antibodies SOX2 Rabbit Abcam-ab92494 1:50 OCT4 MouseMillipore-MAB4401 1:50 Nanog Rabbit Abcam-ab21624 1:50 Alpha smoothmuscle Mouse Sigma Aldrich-A2547 1:500 actin (aSMA) Beta-Tubulin IIIMouse Sigma-T8578 1:200 HNF4-α Rabbit Santa Cruz-SC-8987 1:100 CardiacTroponin T Mouse R&D, MAB1874 1:100 (cINT)-ICC Cardiac Troponin T MouseBD Biosciences-565744 1:50 (cTNT)-Flow Cytometry Alpha GalactosidaseA-ICC Mouse Abnova-H00002717-B01P 1:100 Alpha Galactosidase RabbitAtlas-HPA000237 1:200 A-Western Alpha Galactosidase Rabbit Abnova- 1:100A-PE-Flow A1VMRPSAM001664 GAPDH Rabbit Stem Cell 1:5000Technologies-5174 Globotriaosyicenamide- Mouse BD-551353 1:50 FTTC(Gb3)-ICC Secondary Antibodies Hoechst 33342 Solution NA Thermo-622481:10000 Alexa Fluor555 anti-rabbit Goat Invitrogen-A21428 1:500 AlexaFluor647 anti-rabbit Goat Invitrogen-A-21244 1:500 Alexa Flour488anti-rabbit Goat Invitrogen-A11078 1:500 Alexa Fluor555 anti-mouse GoatInvitrogen-A21422 1:500 Alexa Fluor647 anti-mouse GoatInvitrogen-A-21235 1:500 Alexa Flour488 anti-mouse GoatInvitrogen-A11029 1:500 Horseradish Peroxidase Goat Thermo-31460 1:5000anti-Rabbit IgG (H + L)

Results and Discussion

Derivation and Characterization of Fabry Diseased Induced PluripotentStem Cells

Fabry patient fibroblast were obtained from Coriell Institute, with amutation in the GLA gene (W162X, a well-described pathogenic mutationthat typically results in absent AGA protein activity). Prior toreprogramming, AGA protein levels were detected via Western blot toconfirm disease phenotype. A normal fibroblast line and a recombinanthuman AGA protein were used a, positive controls for AGA protein levels.Fabry diseased fibroblasts exhibited a lack of AGA protein compared towildtype (WT) fibroblasts (FIG. 1 ). Following confirmation of thisdisease characteristic, reprogramming was initiated.

Fabry patient fibroblasts were reprogrammed using the Simplicon RNAReprogramming Kit to create a human Fabry disease cell model.Fibroblasts were transfected with a pluripotent factor containing RNAreplicon and cultured according to the manufacturer's instructions untilsmall stem cell colonies appeared. Induced pluripotent stem cells (iPSC)colonies were clonally expanded and characterized for pluripotenttranscription factor expression. The reprogrammed Fabry iPSC coloniesshowed positive expression and proper localization of three criticalpluripotent transcription factors, Nanog, Oct4 and Sox2 (FIG. 2A, toppanel). In addition to expressing pluripotent proteins, iPSCs must beable to differentiate into each germ layer. Following spontaneousdifferentiation of embryoid bodies formed from Fabry iPSCs, all threegerm layers were detected, ectoderm by β-tubulin III, endoderm by HNF-αand mesoderm by α-smooth muscle actin. To ensure the Fabry iPSCs did notcontain chromosomal abnormalities, which can occur during reprogramming,karyotype analysis was done. No aberrations were detected (FIG. 28 ).

Characterization of Fabry Diseased Induced Pluripotent Stem Cell DerivedCardiomyocytes

Fabry iPSCs were differentiated into cardiomyocytes to develop a modelof Fabry disease in a clinically relevant cell type. Generatedcardiomyocytes began beating around 7 days after differentiation andcontinued to beat until end point analyses (data not shown). Flowcytometry and immunocytochemistry was done on the Fabry iPSCcardiomyocytes to examine purity using a cardiac specific maker, cardiactroponin T (cTNT). The differentiation yielded 97% cTNT positive cells(FIG. 3A), confirmed by immunocytochemistry staining (FIG. 3B).

Transduction with rAAV Carrying the Codon Optimized GLA Gene of SEQ IDNO:1 in Fabry Diseased iPSC-Cardiomyocytes Leads to Robust ProteinExpression of AGA

Fabry iPSC cardiomyocytes were cultured for 14 days post seeding intoexperimental plates prior to transduction of rAAV particles comprising(i) a capsid with a capsid protein of SEQ ID NO.4 and (ii) a nucleicacid comprising a nucleotide sequence of SEQ ID NO:1 operably linked toa CAG promoter of SEQ ID NO:5. Fabry iPSC cardiomyocytes were transducedat MOIs of 25, 100 or 500. The doses were determined from previoustransduction efficiency data using an rAAV comprising (i) a capsid witha capsid protein of SEQ ID NO:4 and (ii) a nucleic acid comprising anEFGP gene operably linked to a CAG promoter. Cells were harvested sixdays post-transduction. Importantly, non-transduced Fabry iPSCcardiomyocytes exhibited a lack of AGA protein by ICC, flow cytometryand Western blot, making them a relevant disease model to examine AGAexpression levels and activity following introduction of the codonoptimized GLA gene of SEQ ID NO:1. Immunocytochemistry (ICC) ofnon-transduced cells showed a lack of AGA protein expression, whereascells transduced with the rAAV exhibited robust AGA staining (FIG. 4A).

Cells were also harvested for flow cytometry and stained for viability,AGA and cTNT. The double positive population was quantified indicatingmature cardiomyocytes (cTNT positive) that had expression of AGA (FIG.4B). A striking dose dependent AGA protein expression response wasobserved following transduction with the rAAV in mature cardiomyocytes.A MOI of only 25 led to 67% cTNT and AGA double positive cells(cTNT+/AGA+) indicating that the rAAV can robustly transduce Fabry iPSCcardiomyocytes and express the codon optimized transgene payload. A MOIof 100 led to 89% cTNT+/AGA+ and a MOI of 250 increased the doublepositive population to 93%.

Total protein was extracted from transduced Fabry iPSC cardiomyocytesfor Western blot to examine total AGA protein in cell lysates.Non-transduced cells lacked AGA protein, an indicator of diseasephenotype. Fabry iPSC cardiomyocytes transduced with the rAAV showedstrong AGA protein expression at the correct size of 49 kDa, afterprobing with an anti-AGA antibody (FIG. 4C).

Enhanced AGA Activity Following rAAV Transduction in Fabry DiseasediPSC-Cardiomyocytes

Transduced Fabry iPSC cardiomyocytes were lysed with alpha-galactosidasebuffer and used to quantify alpha-galactosidase activity with respect toits alpha linkage cleavability. Fabry iPSC cardiomyocyte samples wereincubated with an alpha-galactosidase specific synthetic substrate forone hour to fluorometrically quantify the amount of substrate cleaved.Data revealed a dose dependent increase in AGA activity followingtransduction of an rAAV comprising (i) a capsid with a capsid protein ofSEQ ID NO:4 and (ii) a nucleic acid comprising a nucleotide sequence ofSEQ ID NO:1 operably linked to a CAG promoter of SEQ ID NO:5 in FabryiPSC cardiomyocytes (FIG. 5A).

A hallmark pathology of Fabry disease is cellular Gb3 accumulation(Waldek et al., Life expectancy and cause of death in males and femaleswith Fabry disease: findings from the Fabry Registry. Genetics inMedicine: Official Journal of the American College of Medical Genetics,11(11), 790-796 (2009)). In non-transduced Fabry iPSC cardiomyocytesthere was substantial Gb3 cellular accumulation observed through Gb3immunostaining and fluorescent microscopy (FIG. 5B, top left panel)Transduction of the rAAV at a MOI as low as 25, drastically reduced Gb3accumulation visualized through a decrease in Gb3 immunostaining (FIG.5B).

Conclusion

There is a compelling and urgent need for a durable treatment such as asingle administration intravenous gene therapeutic targeted to keytissues that expresses GLA cell-autonomously, reducing Gb3 and therebyimproving clinical outcomes. The experimental data demonstrates thatdelivery of a codon optimized GLA gene of SEQ ID NO:1 via an AAV with acapsid comprising a capsid protein of SEQ ID NO:4 efficiently targetsFabry disease key organs, particularly cardiac tissue. Transduction ofthis AAV in Fabry diseased iPSC cardiomyocytes resulted in rapid cellautonomous dose-related AGA protein expression and activityintracellularly, well above basal levels. The increase in AGA activityresulted in clearance of Gb3, the accumulation of which is consideredcentral to the pathogenesis of Fabry disease in humans.

Example 3—Codon Optimized GLA cDNA Sequence is Expressed at HigherLevels in Endothelial Cells from Patients with Fabry Disease

Material and Methods

Endothelial Differentiation

Fabry iPSCs were maintained and sub-cultured in 6-well plates every 3-4days prior to dilferentiation. Two 12-well plates were pre-coated withGrowth Factor Reduced Matrigel (Corning). Fabry iPSCs were grown until80-100% confluency in mTESR1. Upon reaching the appropriate density,cultures were subjected to sequential GSK3β and VEGF activation,followed by expansion in StemPro-34 media, following previouslypublished differentiation paradigm (Chalet Meylan, L., Patsch, C., &Thoma, E. (2015). Endothelial cells differentiation from hPSCs. ProtocolExchange. https://doi.org/10.1038/protex.2015.055). On day six,endothelial cells were purified by magnetic cell sorting and were frozenin 5×10⁵ aliquots in CryoStor10 (Stem Cell Technologies). Endothelialcells were thawed at 2.6E+4 cells per cm² onto Fibronectin coated platesin StemPro-34 SFM (1×) media supplemented with VEGF₁₆₅ (Peprotech), 1×Glutamax (Thermo Fisher), and 1× Penicillin/Streptomycin (Gibco15140-122). Endothelial cells were split 1:2 five days after thawingusing Accutase cell detachment solution.

Endothelial Cell Transduction

Endothelial cells were transduced with rAAV comprising (i) a capsid witha capsid protein of SEQ ID NO:4 and (ii) a nucleic acid comprising anucleotide sequence of SEQ ID NO:1 operably linked to a CAG promoterfour days after seeding. Twenty-four hours after transduction, the mediawas aspirated, and fresh StemPro-34 SFM (1×) media was added. Media waschanged every other day. Three wells were harvested to obtain a cellcount. Cells were transduced at multiplicity of infections (MOI) of 50,100, 500, and 1000, based on the cell count and viral titer. Vehicle wasadded such that the volume was equivalent to that of the highest MOI.Cells were incubated for 24 hours and then received a media change. Themedia was changed every other day until harvest, four days posttransduction.

For immunocytochemistry, 10 μM Gb3 (Matreya, LLC.) was added one dayprior, with virus, and to any feedings following transduction to promoteFabry disease-associated accumulation of Gb3.

Immunocytochemistry (ICC)

Fabry iPSC Endothelial ICC

Certain modifications to cell culture were made to enable Gb3 detectionin response to transduction with the rAAV. First, cells were cultured onFibronectin-coated glass chamber slides (LabTek). Cells were loaded with10 μM Gb3, two days prior, upon transduction, and upon washout of virus.Four days post infection, cells were washed once with Phosphate BufferedSaline without magnesium or calcium (PBS−/−) and fixed with 4%paraformaldehyde for one hour at room temperature. Cells were thenwashed 3 times with PBS−/− and blocked for 30 minutes with 2% bovineserum albumin and 5% goat serum in 0.2% Triton X-100 m PBS−/−. Primaryantibody diluted 1:100 against AGA (Abnova H00002717-B01P, customPhycoerythrobilin conjugate) was incubated with cells in blockingsolution for two hours at room temperature. Cells were counterstainedwith DAPI to visualize the nuclei and were imaged on a Zeiss Axiovert.A1inverted microscope. Additionally, conjugated BD clone b5bCD77-Alexa-647 (BD 563632) and Miltenyi CD31-FITC were used byincubating fixed cultures with 1:100 antibody in ICC blocking buffer forone hour.

Western Blot

Four days post-transduction, cells were lifted with Accutase andpelleted at 300×g for 5 minutes. Lysates were made using RIPA buffer(Thermo) plus protease inhibitor cocktail (Roche). Lysates wereincubated on ice for 15 minutes and centrifuged at 21,000×g for 15minutes. Supernatants were collected, and a bicinchoninic acid assay(BCA) was run to determine protein concentration. Concentrations werenormalized. An SDS-PAGE was run using a 4-12% polyacrylamide gel at 200Vfor 30 minutes Protein was transferred to a 0.2 um nitrocellulosemembrane and probed with anti-AGA (Atlas, HPA000237, 1:200) andanti-GAPDH (Stem Cell Technologies, 5174, 1.5000) antibodies followed byspecies specific horseradish peroxidase secondary antibodies overnight.Enhanced chemiluminescence was used to develop protein bands, and banddetection was captured on a BioRad ChemiDoc MP. Band quantification wasanalyzed using Image Lab, Microsoft Excel software and GraphPad Prism8.

AGA Activity Assay

The AGA activity assay was performed with an alpha-galactosidasesynthetic fluorometric substrate (BioVision, K407). AGA activity assaywas performed according to the manufacturer's protocol with thefollowing changes. Briefly, spent media was collected and snap frozenusing liquid nitrogen. Cells were dissociated using trypsin 0.05% andcentrifuged at 300×g for 3 mins to remove supernatant. Cells were thenlysed with AGA buffer and incubated on ice for 10 mins. Samples werecentrifuged at 12,000×g for 10 mins at 4° C. Supernatant was collectedand stored at −80° C. The following day, samples were thawed on ice andprotein quantification via BCA assay was performed in addition to theAGA activity assay. AGA reaction time was 1 hour, samples were read onthe Cytation3 plate reader (BioTek) at 360 excitation/445 emission andgain was set to highest concentration of AGA standard curve.

TABLE 3 list of antibodies Antibody Host Company-Catalog No. DilationPrimary Antibodies CD31-FITC Mouse Miltenyi-130-110-668 1:100 CD144-APCMouse Miltenyi-130-102-738 1:100 GLA-PE Mouse Abnova- H00002717-B01P1:100 CD77-Alexa-647 Mouse BD-563632 1:50

Results

Characterization of Fabry Diseased Induced Pluripotent Stem Cell DerivedEndothelial Cells

Fabry iPSCs were differentiated into endothelial cells to generate arelevant model of a cell type affected in Fabry disease. Generatedendothelial cells were purified by MACS on day 6 for CD144. Flowcytometry and immunocytochemistry was done on the Fabry iPSC endothelialcells to examine purity using an endothelial-specific maker, CD31 Thedifferentiation yielded 99.2% CD31 positive cells (FIG. 6A), confirmedby immunocytochemistry staining at the plasma membrane (FIG. 6B).

Transduction with rAAV Carrying Codon Optimized GLA of SEQ ID NO:1 inFabry Disease iPSC-Endothelial Cells Leads to Robust AGA ProteinExpression

Fabry iPSC endothelial cells were cultured for 11 days from thaw priorto transduction of rAAV comprising (i) a capsid with a capsid protein ofSEQ ID NO:4 and (ii) a nucleic acid comprising a nucleotide sequence ofSEQ ID NO:1 operably linked to a CAG promoter at MOIs of 50, 100, 500,and 1000. The doses were determined from transduction efficiency datausing an otherwise identical rAAV carrying EGFP under the control of CAGpromoter. Cells were harvested four days post-transduction. Flowcytometry showed a dose-dependent increase in AGA-positive cells (FIG.7A) immunocytochemistry (ICC) of non-transduced cells showed a lack ofAGA protein expression, whereas cells transduced with the rAAV exhibitedrobust AGA staining (FIG. 8B).

Protein was extracted from transduced Fabry iPSC-derived endothelialcells for Western blotting to examine total AGA protein. Non-transducedcells lacked AGA protein, as was demonstrated by ICC. However, FabryiPSC endothelial cells transduced with rAAV comprising (i) a capsid witha capsid protein of SEQ ID NO:4 and (ii) a nucleic acid comprising anucleotide sequence of SEQ ID NO:1 operably linked to a CAG promotershowed a strong AGA protein band at the correct size of 49 kDa (FIG.7B), after probing with an anti-AGA antibody. Band density wasdetermined (FIG. 78 ). Transduction with the rAAV significantlyincreased total AGA protein for both MOs compared to non-transducedcells.

Enhanced AGA Activity Following Transduction with rAAV Comprising (i) aCapsid with a Capsid Protein of SEO ID NO:4 and (ii) a Nucleic AcidComprising a Nucleotide Sequence of SEO ID NO:1 Operably Linked to a CAGPromoter in Fabry Diseased iPSC-Endothelial Cells

Transduced Fabry iPSC endothelial cells were lysed with AGA buffer(Biovision, LLC.) and used to quantify AGA activity. Fabry iPSCendothelial cell samples were incubated with an AGA specific syntheticsubstrate for one hour to fluorometrically quantify the amount ofsubstrate cleaved. Data reveals that Fabry iPSC endothelial cellstransduced with the rAAV had enhanced AGA activity compared to FabryiPSC endothelial cells. See FIGS. 8A and 8B.

Conclusion

Transduction of rAAV carrying codon optimized GLA nucleotide sequence ofSEQ ID NO:1 operably linked to a CAG promoter in Fabry diseased iPSCendothelial cells resulted in rapid cell-autonomous, dose-dependent AGAprotein expression and intracellular activity, significantly above basallevels. The increase in AGA activity through expression of the codonoptimized GLA transgene resulted in clearance of Gb3, the accumulationof which is considered central to the pathogenesis of Fabry disease inhumans.

Example 4—Expression and Functional Analysis of AGA Expressed from CodonOptimized GLA of SEQ ID NO:1 in Plasma and Tissues of Fabry Mouse Model

Materials and Methods

In Vivo Study Design and Sample Collection

Fabry model B6;129-Gla^(im/Kul)/J (Jackson Lab #003535) (hereafterreferred toss GLA-null) or normal C57BL/6 mice at 10-11 weeks of agereceived a single intravenous tail vein injection on Day 1 of 1×10¹²,1×10¹³, or 5×10¹³ vg/kg of rAAV comprising (i) a capsid with a capsidprotein of SEQ ID NO:4 and (ii) a nucleic acid comprising a nucleotidesequence of SEQ ID NO:1 operably linked to a CAG promoter in Dulbecco'sPhosphate-Buffered Saline (DPBS) containing 0.005% Pluronic F-68 orvehicle only (refer to Table 4). Mortality, clinical observations, bodyweight, food consumption, bioanalytical analyses for AGA activity andGb3 substrate accumulation, and gross necropsy findings were evaluated.Additionally, tissues from a subset of mice (n=3 per group) wereprocessed and examined for AGA expression and localization byimmunohistochemistry (IHC).

Blood samples (Days 1, 15, and 29) or maximum obtainable (Day 56 only)were collected from all animals via the maxillary vein or the vena cavaafter carbon dioxide inhalation (Day 56 only) for determination ofα-galactosidase A (AGA) activity and/or Gb3 substrate accumulation inplasma. Blood samples were collected, processed to plasma, and stored at−60° C. to −90° C. until the samples were tested. Plasma was analyzedfor AGA activity prior to dosing on Days 1, 15 and 29, and at studytermination on Day 56. Plasma Gb3 and lysoGb3 levels were analyzed onDay 56. Samples of heart, kidney, liver, and small intestine were alsocollected at study termination and analyzed for AGA activity and Gb3substrate level. Representative samples of select tissues (heart, liver,kidney, and small intestine) from a subset of animals in each group werecollected and immediately preserved in 10% neutral buffered formalin(20-30× volume of the tissue). Tissues were fixed for approximately 48hours (±1 hour) at room temperature and then transferred to 70% ethanol.

TABLE 4 Scheme of Treatment Groups for Fabry Mouse Dose-Ranging a ndEfficacy Study Group Genotype rAAV (vg/kg) N 1 C57BL/6 Vehicle 15 2GLA-null Vehicle 15 3 GLA-null 1 × 10¹² 15 4 GLA-null 1 × 10¹³ 15 5GLA-null 5 × 10¹³ 15

Plasma and Tissue Activity Analysis

AGA enzyme activity was detected and quantified via the production of4-methylumbelliferone using a validated assay. Briefly, samples werediluted in Activity Buffer containing4-Methylumbelliferyl-α-D-galactopyranoside and incubated for 1 hour atroom temperature. Fluorescence emission was quantified using a platereader equipped with fluorescence detection at 366 excitation/450-475emission and compared to a standard curve. Total protein within tissuesamples was quantified using a BCA assay.

Plasma Substrate Analysis

LysoGb3 (m/z 786) and its analogs (m/z 784, m/z 802, and n/z 804), andGb3 and its isoforms (C16:0, C18:0, C22:0, C24:1, and C24:0) in plasmawere analyzed as described (Boutin & Auray-Blais, 2014, AnalyticalChemistry, 86(7), 3476-3483. //doi.org/10.1021/ac-404000d; Provencal etal. 2016, Bioanalysis, 8(17), 1793-1807.https://doi.org/10.4135/bio-2016-0116). Briefly, plasma samples drawn 56days post-dose (42 day samples were saved and archived but not analyzed)were spiked with glycinated-lysoGb3 (Matreya LLC; State College, PA) asthe internal standard and purified by solid phase extraction usingmixed-mode strong cation exchange (MCX) cartridges (Waters Corporation,Milford, MA). The collected phase was dried under a nitrogen stream,reconstituted in 50% acetonitrile/0.1% formic acid, and injected onto anAcquity 1-Class ultra performance liquid chromatography (UPLC) system(Waters Corporation, Milford, MA) using an BEH C18 column and lysoGb3was detected simultaneously with the spiked-in internal standards(glycinated-lysoGb3) using a Xevo TQ-S (Waters Corporation) triplequadrupole tandem mass spectrometer.

For Gb3 analysis, the plasma samples were spiked with Gb3 (C18:0D3)(Matreya LLC; State College, PA) as the internal standard and purifiedby liquid-liquid extraction with tert-butyl methyl ether. Samples weresaponified with potassium hydroxide, neutralized with acetic acid,centrifuged, and the organic layer collected and dried under a stream ofnitrogen. The sample was reconstituted and injected onto an AllianceHPLC 2795 system (Waters Corporation, Milford, MA) using a ZorbaxBonus-RP Guard column cartridge (4.6×12.5 mm, Agilent Technologies).Analytes were then detected simultaneously with the spiked-in internalstandards using a Quattro micro triple quadrupole tandem massspectrometer (Waters Corporation).

Tissue Substrate Analysis

Gb3 isoforms and analogs (C16:0, C18.0, C20:0, C22:0, and C24:1 intissues important for the treatment of Fabry disease were analyzed asdescribed (Provencal et al. 2016, Bioanalysis, 8(17), 1793-1807.https://doi.org/10.4155/bio-2016-0116) Briefly, frozen heart, kidney,liver, small intestine, and spleen tissue samples in reinforced tubeswere thawed, five ceramic beads added to each tube, and the sampleshomogenized in methanol using a Bead Disruptor 12 bead mill homogenizer(Omni International; Kennesaw, GA). Lysates were then extracted andanalyzed in the same manner as the plasma samples, described above. Alltissues were collected from n=12 animals per group, except for thehearts from n=6 animals per group due to tissue size limitations toenable AGA activity analysis from the other six animals. The remainingthree animals from each treatment group were used forimmunohistochemistry analysis.

Data are represented as a response ratio, which is the ratio of the massspectrometer peak area of the sample analyte divided by the peak area ofthe Gb3 internal standard (Gb3 (C18:0D₃)). For each tissue, the totalresponse ratios of all six Gb3 isoforms/analogs was examined. Because ofthe nature of this ratio, there are no units ascribed to this ResponseRatio.

Statistical Analysis

Statistical analysis was performed by the biostatistics group at CharlesRiver Laboratories—Mattawan (Mattawan, MO). Data analysis was performedon the aggregate values of all lysoGb3 and Gb3 isoforms/analogs toensure that the data were capturing a broad spectrum of AGA substrates.All “not detected” (N.D.) values in both the plasma lysoGb3 and thetissue Gb3 data sets were treated as having values of zero for thepurposes of data analysis. A sensitivity analysis was performedsubstituting LLOD (lower limit of detection) values for those N.D.values.

Tests to assess homogeneity of group variances and the normality of theresiduals were performed at the 0.01 level of significance. Theexperimental unit used for the Gb3 data analysis was the individualanimal.

The raw data was tabulated within each time interval, and the mean andstandard deviation calculated for each endpoint by group. For eachendpoint, treatment groups were compared to the control group using theanalysis outlined below. Data for some endpoints, as appropriate, weretransformed by either a log or rank transformation prior to conductingthe specified analysis.

TABLE 5 Statistical Comparisons Calculated Statistical Comparisons GroupVersus Groups 1 2, 3, 4, 5 2 3, 4, 5

For endpoints and/or parameters (within each collection interval) thatdemonstrate variability, and where sample sizes for all groups are threeor greater, the system tested the normality of the residuals andhomogeneity of variances to see whether the data was approximatelynormal or whether a log transformation or rank transformation should beused. Levene's test was used to assess homogeneity of group variancesand Shapiro-Wilk's test was used to test the normality of the residuals.

On the raw data, if Levene's test was not significant (p≥0.01) andShapiro-Wilk's test was not significant (p≥0.01), then a normaldistribution was used. If either the Levene's test was significant(p<0.0l) or Shapiro-Wilk's test was significant (p<0.01), normality andhomogeneity of variances were tested with a log transformation used onthe data.

On the log transformed data, if Levene's test was not significant(p≥0.01) and Shapiro-Wilk's test was not significant (p≥0.01), then alog normal distribution was used. If either the Levene's test wassignificant (p<0.01) or Shapiro-Wilk's test was significant (p<0.01),then a rank transformation was used on the data.

A one-way analysis of variance using the appropriate transformed datawas used to test each endpoint for the effects of treatment (Edwards &Berry, 1987, Biometrics, 43(4), 913-928).

If the treatment effect was significant (p<0.05), linear contrasts wasconstructed for pair-wise comparison of treatment groups as describedabove. Results of these pair-wise comparisons were reported at the 0.05and 0.01 significance levels after adjustment for multiple comparisonsusing the methods of Edwards and Berry (Edwards & Berry, 1987,Biometrics, 43(4), 913-928). All tests were two-tailed tests unlessindicated otherwise.

Results and Discussion

Plasma and Tissue AGA Analysis

A single IV dose of rAAV as described above (comprising (i) a capsidwith a capsid protein of SEQ ID NO:4 and (ii) a nucleic acid comprisinga nucleotide sequence of SEQ ID NO:1 operably linked to a CAG promoter)resulted in consistent and dose-dependent increases in plasma AGAactivity at every evaluated timepoint (FIG. 9A). These increases weremaintained over the eight-week duration of the study (FIG. 9A).

At study termination, an increase in AGA activity was detected in thekidney and small intestine compared to vehicle-treated mice, with higherlevels noted in the heart at the high dose of 5×10¹³ vg/kg (FIG. 9B). Inthe liver, high AGA activity levels were observed in mice treated withboth 1×10¹³ and 5×10¹³ vg/kg (FIG. 9B). These data suggest that a singleIV dose of rAAV as described above (comprising (i) a capsid with acapsid protein of SEQ ID NO:4 and (ii) a nucleic acid comprising anucleotide sequence of SEQ ID NO:1 operably linked to a CAG promoter)results in significant expression of AGA activity in plasma particularlyat doses of 1×10¹³ and higher. Significant expression was also seen outto eight weeks post-dose in the heart and liver. To visually confirm AGAexpression in tissues, heart, kidney, small intestine, and liver werecollected, fixed, paraffin-embedded, and subjected to IHC for AGA asdescribed above. A dose-dependent increase in AGA expression wasobserved in all tissues examined (FIG. 10 ).

Plasma LysoGb3 Analysis

A single IV dose of rAAV as described above (comprising (i) a capsidwith a capsid protein of SEQ ID NO:4 and (ii) a nucleic acid comprisinga nucleotide sequence of SEQ ID NO:1 operably linked to a CAG promoter)resulted in a dose-dependent and significant (P<0.01) reduction at alllevels of total lysoGb3 levels in plasma eight weeks after dosing. Fourdifferent analogs of lysoGb3 were examined, represented by theirmass-to-charge ratios (m/z): m/z 786, m/z 784, m/802, and m/z 804.Examining the aggregate change of all analogs together, lysoGb3 wassubstantially reduced at all dose levels of the rAAV.

Whereas wildtype mice exhibit only 2.11±0.26 nM of lysoGb3 detected inthe plasma, vehicle-treated Fabry mice have a significant lysoGb3accumulation in plasma of more than 477.37±88.5 nM (FIG. 11 ). A singlelow IV dose of 1×10¹² vg/kg of the rAAV reduced the concentration oflysoGb3 in plasma of Fabry mice by more than 58%, to 197.5±124.5 nM(P<0.01). The mid dose of 1×10¹³ vg/kg of the rAAV resulted in a 96%reduction of plasma lysoGb3 in Fabry mice, to 19.1±13.46 nM (P<0.01).Further increasing the dose of the rAAV to 5×10¹³ vg/kg resulted in asimilar reduction in lysoGb3 to 10.57±13.85 nM (P<0.01). Data from theaggregated change of all analogs are summarized in Table 6 below:

TABLE 6 Plasma lysoGb3 Levels at Day 56 Post-IV Injection Mean Controlof All LSMEAN LysoGb3 minus P Value Dose Analogs Treatment comparedSignificant Group Genotype Treatment (vg/kg) (nM) LSMEAN to Group 2 At 1WT Vehicle N/A   2.11 ± 0.26  N/A N/A N/A 2 GLA-null Vehicle N/A 477.37± 88.5  N/A N/A N/A 3 GLA-null rAAV 1 × 10¹²  197.5 ± 124.5 −13.39050.0000 <0.01 4 GLA-null rAAV 1 × 10¹³   19.1 ± 13.46 −30.5238 0.0000<0.01 5 GLA-null rAAV 1 × 10¹³  10.57 ± 13.85 −42.0571 0.0000 <0.01

Gb3 and Gb3 analogs were also examined in plasma but only lysoGb3 dataare discussed here because in plasma lysoGb3 is a more relevant form ofsubstrate for the clinical disease (Boutin & Auray-Blais, 2014,Analytical Chemistry, 86(7), 3476-3483. /doi.org/10.1021/ac404000d).

Tissue Gb3 Analysis

All tissues examined exhibit significantly greater amounts of Gb3 inGLA-null Fabry mice relative to wildtype C57BL/6, confirming theexpected accumulation of AGA substrate associated with the Fabry diseasephenotype. In all tissues and at all dose levels, there aredose-dependent reductions of Gb3 following treatment with a single IVdose of the rAAV. Specific results from heart, kidney, liver, and smallintestine are described below. Spleen was also collected and analyzed,but those results are not discussed here since spleen is not of clinicalsignificance to Fabry disease. Tissue Gb3 data are represented as aresponse ratio, which is the ratio of the mass spectrometer peak ara ofthe sample analyte divided by the peak ara of the Gb3 internal standard(Gb3 (C18:0D₃)).

Heart Gb3 Analysis

Whereas wildtype C57BL/6 mice exhibit response ratio of 2.9±0.2 in theheart, GLA-null Fabry mice show a significant substrate accumulationmeasured at 285.5±56.5 (P<0.01). A single IV injection of the rAAVresults in a dose-dependent clearance of Gb3 in the Fabry mice (FIG. 12). The low dose of 1×10¹² vg/kg reduced the Gb3 content of the heart by89.4%, to 30.1±30.5. The mid dose level of rAAV of 1×10¹³ vg/kg reducedthe Gb3 level by more than 96%, to 9.9*3.7. The high dose of 5×10¹³vg/kg cleared more than 98% of Gb3, to 4.4±2.2. These results representan average of n=6 animals per group. All Gb3 reductions in heart werestatistically significant (P<0.01) in comparison to Group 2(vehicle-treated GLA-null).

Kidney Gb3 Analysis

Vehicle-treated wildtype C57BL/6 mice show response ratio of 25±4.1whereas the GLA-null Fabry mice exhibit 373±71. A single TV dose of therAAV resulted in a dose-dependent and statistically significant (P<0.01)clearance of Gb3, exhibiting a 28.3%, 74.4%, and 90.7% reduction 56 daysafter treatment with 1×10¹², 1×10¹³, or 5×10¹³ vg/kg, respectively. Gb3reductions in kidney at the mid and high dose levels were statisticallysignificant (P<0.01) in comparison to Group 2 (vehicle-treatedGLA-null). The low dose 1×10¹² vg/kg did not achieve significance(P=0.0537) although the therapeutic product does exhibit a trendingreduction in Gb3 even at this low dose level (FIG. 12 ).

Liver Gb3 Analysis

The Gb3 levels, measured in Response Ratio, in liver lysates of wildtypeC57BL/6 and GLA-null Fabry mice were 2.9±0.5 and 377±75, respectively.Liver Gb3 accumulation was almost completely cleared by even the lowdose of the rAAV (FIG. 12 ). The 1×10¹² vg/kg dose resulted in a 93.1%reduction of Gb3, to 26±24. The mid and high doses of 1×10¹³ or 5×10¹³vg/kg resulted in nearly complete (>99%) substrate clearance, revertingback to wildtype levels of 2.6±0.5 and 2.5±0.3, respectively.

Small Intestine Gb3 Analysis

Small intestine showed a much higher accumulation of Gb3 in the GLA-nullFabry mice, exhibiting response ratio of 1164±276, whereas only 3.4±0.3was detected in the small intestine of wildtype C57BL/6 mice. A single1V dose of the rAAV resulted in a dose-dependent and significant(P<0.01) at all dose levels relative to vehicle-treated GLA-nullclearance (FIG. 12 ). 1×10¹², 1×10¹³, or 5×10¹³ vg/kg dose levelsresulted in 33.6% (772±148), 73% (314±190), and 94.8% (60±59) reductionin Gb3, respectively.

TABLE 7 Summary of Gb3 Reduction in Tissues Collected 56 Days After IVTreatment with rAAV carrying codon optimized GLA Response Ratio %Reduction from of Mean Total Vehicle-Treated Group Genotype Dose (vg/kg)Tissue Gb3 Content GLA-Null 1 WT Vehicle Heart 2.9 N/A 2 GLA-nullVehicle Heart 285.5   0% 3 GLA-null 1 × 10¹² Heart 30.1 89.4% 4 GLA-null1 × 10¹³ Heart 9.9 96.5% 5 GLA-null 5 × 10¹³ Heart 4.4 98.5% 1 WTVehicle Kidney 25.0 N/A 2 GLA-null Vehicle Kidney 373.1   0% 3 GLA-null1 × 10¹² Kidney 267.4  28.3%* 4 GLA-null 1 × 10¹³ Kidney 95.5 74.4% 5GLA-null 5 × 10¹³ Kidney 34.6 90.7% 1 WT Vehicle Small Intestine 3.4 N/A2 GLA-null Vehicle Staal Intestine 1164.2 0% 3 GLA-null 1 × 10¹² SmallIntestine 772.7 33.6% 4 GLA-null 1 × 10¹³ Small Intestine 313.8 73.0% 5GLA-null 5 × 10¹³ Small Intestine 60.5 94.8% 1 WT Vehicle Liver 2.9 N/A2 GLA-null Vehicle Liver 377.3   0% 3 GLA-null 1 × 10¹² Liver 25.9 93.1%4 GLA-null 1 × 10¹³ Liver 2.6 99.3% 5 GLA-null 5 × 10¹³ Liver 2.5 99.3%

Conclusions

Fabry disease is caused by the accumulation of Gb3 and lysoGb3 substratemolecules in organs and tissues. This study evaluated the ability of asingle dose of rAAV carrying codon optimized GLA of SEQ ID NO:1 toexpress AGA and catabolize Gb:3 as a treatment for Fabry disease in amouse model. The results of this study demonstrate significantreductions in Gb3 and lysoGb3 in plasma and all tissues examined. Thelow dose of 1×10¹³ vg/kg is highly active in both the heart and liver,leading to a greater than 89% substrate reduction in each organ. The lowdose produced a 33.4% substrate reduction in the small intestine, and areduction in the kidney although the variability seen in the data in thekidney was such that that result did not achieve statisticalsignificance. The mid dose of 1×10¹³ vg/kg yielded more than 96%substrate reduction in the heart, nearly complete (>99%) clearance ofthe liver, 74.4% reduction in the kidney and 73% reduction of the smallintestine and this is a highly active and significant dose in alltissues tested. The high dose of 5×10¹³ vg/kg resulted in greater than98% substrate clearance in the heart, more than 90% clearance in thekidney, almost 95% clearance in the small intestine, and more than 99%clearance in the liver. All of the doses tested am active andefficacious in the Fabry mouse model and reduced accumulated AGAsubstrate levels to in some cases at or near wildtype levels.

Example 5—Expression and Functional Analysis of AGA Expressed from CodonOptimized GLA of SEQ ID NO:1 in Plasma and Tissues of Wild Type Mice

A single dose non-GLP acute tolerability study of rAAV comprising (i) acapsid with a capsid protein of SEQ ID NO:4 and (ii) a nucleic acid ofSEQ ID NO:6, comprising codon-optimized GLA of SEQ ID NO:1 operablylinked to a CAG promoter (4D-310) by intravenous injection inexperimentally naive C57BL/6 male mice (8 weeks in age at initiation ofdosing) was performed. The maximum tolerated dose (MTD) following asingle IV injection (via tail vein) was determined.

The study design is provided at Table 8 below:

TABLE 8 Dose Level Dose Volume No. of Male Group No. (vg/kg) (mL/kg)Animals 1 0 10 6 2 1.0 × 10¹³ 10 6 3 5.0 × 10¹³ 10 6 4 1.5 × 10¹⁴ 10 6

Blood samples were collected on Day 1 (predose) and at the same time ofday on Days 15, 29 and 43 from all surviving animals via maxillaryvessel for determination of GLA activity in plasma.

Postmortem study evaluations were performed on 1 animal euthanized onDay 30 and all surviving animals at the scheduled terminal necropsy (˜6weeks post dose). Heart, liver (left lateral lobe), kidney (left) andsmall intestine (comparable sized sections of duodenum, ileum andjejunum) were isolated from all animals and evaluated for GLA activity.

Following dosing, all treated plasma samples GLA activity significantlyabove that of control animals (see FIG. 13 , illustrating a 1200-foldincrease over vehicle control for the 1.0×10¹³ vg/kg dose, a 5000-foldincrease over vehicle control for the 5.0×10¹³ vg/kg dose and a26,000-fold increase over vehicle control for the 1.5×10¹³ vg/kg dose).In addition, each higher dose had plasma GLA activity significantlyabove the lower dose(s). The same trends (dose responsiveness) wereobserved in all collected tissues (see FIG. 14 ). Taken collectively,the data demonstrate a clear expression of GLA and resultant GLAactivity following a single IV dose of 4D-310 at each dose. Notably,duodenum, heart, ileum, jejunum, heart kidney, and liver tissues areamong the most relevant tissues for treatment of Fabry disease, withexpression of GLA in the liver serving as a GLA bioreactor.

A summary of GLA values in the plasma of 4D-310-treated animals isprovided in Table 9 below:

TABLE 9 Dose Sample (vg/kg) Type Day 1 Day 15 Day 29 Day 43 (plasma,nmol/hr/mL) 0 Plasma  85.18   114.33   109.33   100.72 1.0 × 10¹³ 113.43 134116.67^(b)  148983.33^(b)  136833.33^(b) 5.0 × 10¹³ 100.43 549500.00^(b,d)  600000.00^(b,d)  637000.00^(b,d) 1.5 × 10¹⁴  91.033211666.67^(b,d,f) 2980000.00^(b,d,f) 3023333.33^(b,d,f) (tissues,nmol/hr/mg) 0 Duodenum — — —   397.67 1.0 × 10¹³ — — —   1646.33^(b,)5.0 × 10¹³ — — —   4188.00^(b,d) 1.5 × 10¹⁴ — — —  27883.33^(b,d,f) 0Heart — — —   146.67 1.0 × 10¹³ — — —   6246.67^(b) 5.0 × 10¹³ — — — 24380.00^(b,d) 1.5 × 10¹⁴ — — —  71833.33^(b,d,f) 0 Ileum — — —  432.75 1.0 × 10¹³ — — —   2253.33^(b) 5.0 × 10¹³ — — —  13450.00^(b,d)1.5 × 10¹⁴ — — —  39466.67^(b,d,f) 0 Jejunum — — —   366.33 1.0 × 10¹³ —— —   2471.50^(b) 5.0 × 10¹³ — — —   6564.00^(b,c) 1.5 × 10¹⁴ — — — 26783.33^(b,d,f) 0 Kidney — — —   132.67 1.0 × 10¹³ — — —   1389.17^(b)5.0 × 10¹³ — — —   3828.00^(b,d) 1.5 × 10¹⁴ — — —  12356.67^(b,d,f) 0Liver — — —   282.00 1.0 × 10¹³ — — —  86816.67^(b) 5.0 × 10¹³ — — —2020000.00^(b,d) 1.5 × 10¹⁴ — — —  401333.33^(b,d,f) ~Not Applicable^(b) = different from 0 vg/kg, p < 0.01 ^(c) = different from 1.0 × 10¹³vg/kg p < 0.05 ^(d) = different from 1.0 × 10¹³ vg/kg p < 0.01 ^(f) =different from 5.0 × 10¹⁴ vg/kg p < 0.01

There were no 4D-310-related macroscopic observations in the study.There were no 4D-310-related clinical observations of systemic or localtoxicity in the study, nor were any 4D-310-related changes in bodyweight or effects on food consumption observed. There were no 4D-310related mortalities in the study.

As there were no adverse 4D-310 related effects on any of the evaluatedparameters following a single intravenous dose at 1.0×10¹³, 5×10¹³ and1.5×10¹⁴ vg/kg to male mice, the no-observed-effect-level (NOEL) andmaximum tolerated dose was determined to be 1.5×10¹⁴ vg/kg, the highestdose tested.

Example 6—Toxicity and Biodistribution of Systemically Administered4D-310 (Comprising a Nucleic Acid with Codon Optimized GLA of SEQ IDNO:1)

Potential toxicity and tissue biodistribution of 4D-310, administered toC57BL/6 (wt) mice via a single intravenous injection via tail veinfollowed by a 14-day and 91- or 92-day observation period wasinvestigated. The study design is provided in Table 10 below:

TABLE 10 No. of Male Animals Dose Level Dose Volume Day 15 Day 92 +/− 1Group No. (vg/kg) (ml/kg) Necropsyª Necropsy^(a) 1 0 10 22 + 2^(b) 22 +2^(b) 2 1.0 × 10¹³ 10 22 + 2^(b) 22 + 2^(b) 3 5.0 × 10¹³ 10 22 + 2^(b)22 + 2^(b) 4 1.5 × 10¹⁴ 10 22 + 2^(b) 22 + 2^(b) ^(a)On each day ofnecropsy, animals were designated as follows: 5/group for clinicalchemistry parameters and microscopic examinations; 5/group forhematology parameters; 5/group for anti-capsid and anti-payloadanti-drug antibody analyses; 7/group for GLA activity analysis andtissue and whole blood qPCR and RT-qPCR. ^(b)Additionalanimals/treatment group were dosed as possible replacements. If not usedas replacements, the additional animals were euthanized and discarded atthe Day 92 ± 1 interval

The following parameters and endpoints were evaluated in this study:mortality, clinical observations, body weight, and food consumption,ophthalmoscopic examinations, clinical pathology parameters (hematologyand clinical chemistry), anti-capsid and anti-payload antidrug antibodyanalyses, GLA activity analysis, biodistribution analysis (tissue andwhole blood qPCR and RT-qPCR), gross necropsy findings, organ weights,and histopathologic examinations.

There were no 4D-310-related mortalities or clinical signs of systemicor direct local toxicity, nor any effects on body weight, foodconsumption, ophthalmoscopic examinations, clinical pathologyparameters, organ weights, or gross necropsy findings.

All 4D-310 (which comprises the 4D-C102 capsid protein of SEQ ID NO:4)treated animals screened positive for anti-4D-C102 capsid antibodiesbased on being above the determined screening cut-point. However, eventhough all 4D-310 treated animals screened positive; only 16/30 sampleswere confirmed positive following the evaluation against theconfirmatory cut-point. These 16 confirmed positive samples had thefollowing dose level breakdown: 7/10 at 1.0×10¹³ vg/kg, 5/10 at 5.0×10¹³vg/kg, and 4/10 at 1.5×10¹⁴ vg/kg. There was no apparent doserelationship to the anti-capsid antibody response

A proportion of animals developed anti-capsid antibodies in the studyfollowing dosing with 4D-310, however there was no dose-relationshipapparent. All control animals were screened and confirmed to be negativefor anti-4D-C102 capsid antibodies. 4D-310 treatment did not result inanti-alpha Galactosidase A antibodies being formed as all treatedanimals were found to be negative. Treatment with 4D-310 yielded a dosedependent and statistically significant increase in plasma GLA activitythat was present throughout the duration of the study when compared tothe control group. Most treated groups were also higher than theprevious dose group in a statistically significant manner.

Among the highest levels of vector genomes observed for both doses andtimepoints are heart, liver, lung, kidney and injection site. Theconcentration of 4D-310 vector DNA detected in the Brain and Spinal Cordin all groups was overall lower compared to the previously mentionedtissues. Similar to the biodistribution analysis, where the highestlevels of 4D-310 vector DNA was detected in the liver samples, vectorderived gene expression was observed at the highest concentrations inthe liver samples, followed by the heart, lung, and injection sitesamples.

Within the liver samples, 4D-310 expression was approximately 14×greater at 645,293,620 copies per μg of total RNA compared to the heartsamples with at 44,662,881 copies per μg of total RNA. The testissamples exhibited an abnormally increased level of gene expression at anaverage of 2,888,001 copies per μg of total RNA compared only 1,884copies of 4D-310 vector DNA detected in the biodistribution analysis atthe same timepoint and dose level. This can be attributed directly toAnimal 4D-310, where 4D-310 expression was 15,095,299 copies versus anaverage of only 446,541 copies between the other 5 animals. In addition,Animal 4043 only had an individual count of 1,443 copies of 4D-310vector DNA detected during biodistribution analysis and had noclinical/veterinary findings throughout the duration of the study.Therefore, the abnormally high level of gene expression was considerednon-adverse as there was no impact to the overall health of the animal.

Overall vector-derived 4D-310 gene expression was noted in all tissueswith the highest concentrations noted in the liver, heart, lung, andinjection site samples. There were no 4D-310 macroscopic findings. Therewere no 4D-310-related microscopic findings with the exception ofminimal to mild injection site perivascular/vascular inflammation at≥5.0×10¹³ vg/kg. This finding was not present in the Day 92±1 animalsand was considered reversible.

A summary of the mean copies per μg of tissue vector DNA is summarizedin Table 11 below.

TABLE 1B Low Dose - High Dose - High Dose Day - TISSUE Vehicle - Day 15Day15 Day15 92/93 Brain, Cerebrum 0 2,886.20 21,176.88 17,799.26 Brain,Cerebellum 29.56 2,657.93 13,488.98 18,107.84 Dorsal Root Ganglia 0709.87 6,138.45 1,847.41 (DRG) Epididymis 0 521.96 7,207.75 2,930.27Heart 8.38 19,243.03 81,560.75 61,892.67 Injection Site 0 224,576.423,989,305.62 850,106.33 Kidney Left 0 6,932.66 85,867.02 28,735.23 Liver0 425,413.55 6,798,453.78 3,379,680.04 Lung with Bronchi 0 85,046.08340,084.19 246,525.53 Lymph Node, 0 1,703.21 12,431.13 3,621.43Mandibular Lymph Node, 0 1,786.74 13,209.12 4,528.63 Mesenteric SciaticNerve 0 1,304.78 15,593.00 6,724.72 Seminal Vesicles Left 0 240.721,845.50 1,149.30 Spinal Cord 429.92 2,888.00 22,941.23 21,961.64 Spleen0 16,851.98 237,296.48 10,989.76 Testis 7.14 508.36 4,745.76 1,884.38Whole Blood 0   208,234.43 146.80 Bone Marrow 0 140.94 5,641.30 790.94BLOD = Below Limit of Quantitation, 9.97 Copies/reaction BLOQ = BelowLower Limit of Quantitation, 50 copies/reaction N/A = Total DNAConcentration (μg/μL) is ≤0 — = No samples available for analysis. Note:Individual BLOD results were reported as “0” and Individual BLOQ resultswere reported as “50” for the calculation of the mean copy number; N/Aresults are not included in the mean copy number

Similar to the biodistribution analysis, where the highest levels of4D-310 vector DNA was detected in the liver samples, vector derived geneexpression was observed at the highest concentrations in the liversamples, followed by the heart, lung, and injection site samples. Withinthe liver samples, 4D-310 expression was approximately 14% greater at645,293,620 copies per μg of total RNA compared to the heart sampleswith at 44,662,981 copies per μg of total RNA.

Overall vector-derived 4D-310 gene expression was noted in all tissueswith the highest concentrations noted in the liver, heart, lung, andinjection site samples. A summary of the mean copies per μg of tissuevector RNA is summarized in Table 12 below.

TABLE 12 TISSUE High Dose Day - 92/93 Brain, Cerebrum 913,078.68 Brain,Cerebellum 741,324.07 Dorsal Root Ganglia (DRG) N/A Heart 44,662,881.13Injection Site 12,798,244.57 Kidney 278,022.32 Liver 645,293,620.04 Lungwith Bronchi 15,668,829.48 Spinal Cord 252,581.65 Spleen 510,369.25Testis 2,888,000.80 BLOD = Below Limit of Quantitation, 57.34copies/reaction BLOQ = Below Lower Limit of Quantitation, 500copies/reaction N/A = Total RNA Concentration (μg/μL) is ≤0 Note:Individual BLOD results were reported as “0” and Individual BLOQ resultswere reported as “500” for the calculation of the mean copy number; N/Aresults are not included in the mean copy number

In conclusion, following a single intravenous administration of 4D-310,no adverse effects were noted in any parameter evaluated. As a result,the No-Observed-Adverse-Effect-Level (NOAEL) for local and systemictoxicity was 1.5×10¹⁴ vg/kg, the highest dose level tested.

Example 7—Study on IV Dosing of 4D-310 to Non-Human Primates

Toxicity, pharmacodynamics and biodistribution of 4D-310 and4D-C102.CAG-EGFP (an rAAV having the same capsid as 4D-310 and aheterologous nucleic acid encoding EGFP operably linked to a CAGpromoter) was assessed in cynomolgus monkeys following a singleintravenous (infusion) dose prior to human clinical trials. The studydesign is shown in Table 13 below:

TABLE 13 Dose In- Immune Cohort Group N Treatment Route (vg/kg) LifeSuppression Endpoints 1 2 3 4D-310 IV 3e12 8 wks Drug: In-Life 3 34D-310 IV 1e13 8 wks Methylprednisolone Cageside 4 3 4D-310 IV 5e13 8wks Dosing: 1M q.w. observations (2x 2 1 1 Vehicle IV N/A 8 wks DoseLevels: daily) 5 3 C102.EG IV 5e13 8 wks 40 mg/kg: Day15 Feedconsumption EP to 28 (2x daily) 20 mg/kg: Day 29 Detailed Clinical to 42Observations 10 mg/kg: Day 43 (weekly) to 56 Body weights (weekly)Hematology (Week 1, 2, 4, 6, EOS) Clinical chem (Week 1, 2, 4, 6, EOS)Plasma AGA activity (pre-dose, Week 2, 4, 6 EOS) After sacrifice:Tissues (primary): Heart Liver Kidney Small Intestines AGA activityHistopathology Immunochemistry for AGA qPCR (biodistribution) RT-qPCR(transgene expression)

Cynomolgus monkeys were administered either of two different transgenes:GLA (of SEQ ID NO:1) (Groups 2-4) or EGFP (Group 5), each comprisedwithin an rAAV capsid comprising a capsid protein of SEQ ID NO:4, orsingle vehicle control (Group 1). A remarkably clean safety profile wasobserved with 4D-310 (comprising the codon optimized GLA of SEQ ID NO:1and capsid of SEQ ID NO:4)-treated animals. Transaminitis was observedonly in EGFP-dosed animals.

All animals were administered an immunosuppressant beginning two weeksprior to test article administration through study termination. Thissuppression regimen was intended to be aligned with clinical practice.Briefly, from Day −15 through Day 28, methylprednisolone acetate (40mg/kg) was administered once weekly via intramuscular injection to allanimals in Groups 2, 3, and 4. The dose was halved to 20 mg/kg on Days29 through 42 and halved again to 10 mg/kg on Days 43 through 56.Immunosuppression dosing was not required the week of scheduled terminalnecropsy. From Day −14 through 29, methylprednisolone acetate (40 mg/kg)was administered once weekly via IM injection to all animals in Groups 1and 5. Immunosuppression was increased (to a max of 80 mg/kg/week) ordecreased at the discretion of the study veterinarian. On Day 16, oneGroup 5 male received an additional dose (40 mg/kg).

The vehicle and test articles were administered once during the studyvia IV infusion at dose levels of 0 (vehicle), 3.0×10¹², 1.0×10¹³,5.0×10¹³ vg/kg for 4D-310 and 5.0×10¹³ vg/kg for 4D-C102.CAG.EGFP andadministered at a rate of 3 mL/minute. The first day of dosing wasdesignated as Day 1 for each individual animal.

The IV route is the intended route of administration of 4D-310 in humansubjects. The dose levels were selected based on separate dose rangingsafety/efficacy studies in mice in which doses from 1×10¹² vg/kg to1.5×10¹⁴ vg/kg were all shown to be safe.

For GLA plasma sample collection, blood samples (approximately 3 mL)were collected from all animals via the femoral vein for GLA analysisaccording to the schedule at Table 14 below:

TABLE 14 Sample Collection Time Points Day 1 Day of Group No. (predose)Day 15 Day 29 Day 43 Necropsy 1-4 X X X X X X = sample collected

For anti-AAV evaluation, blood samples (approximately 5 mL) werecollected from all animals via femoral vein according to the schedule atTable 15 below:

TABLE 15 Sample Collection Time Points Group Day 1 Day of No. (predose)Day 8 Day 15 Day 29 Day 43 Necropsy 1-4 X X X X X X X = sample collected

Tissue samples for qPCR analysis (100 to 180 mg for each tissue sampleexcept spleen (50-80 mg/sample) were collected and split into 2 samplesand snap-frozen liquid nitrogen and stored frozen until analyzed. DNAtissue collection and evaluation was according to Table 16 below:

TABLE 16 Groups 2-4 Group 1 Group 5 Tissue^(a) Region DNA 1 DNA 1 DNA 2(GLA) (GLA) (GFP) Blood Aorta 1 1 1 vessel Carotid 1 1 1 Pulmonary — — 1Artery Brain^(b) Left 1 1 1 Hemisphere Right 1 1 1 Hemisphere Brain Stem1 1 1 Cerebellum 1 1 1 Dorsal Thoracic — — — Root All remaining 1 1 1Ganglia (L) All remaining — — — (R) Nerve Sciatic Nerve 1 1 1 Heart Left2 2 2 Ventricular Free Wall Left Atrium 1 1 1 Right Ventricle 1 1 1Right Atrium 1 1 1 Ventricular 1 1 1 Septum Kidney Right Kidney 1 1 1Left Kidney 1 1 1 Liver Right Lobe 1 1 1 Left Lobe 1 1 1 Lung 2 2 2Skeletal Deltoid (p) 1 1 1 Muscle Deltoid (d) 1 1 1 Diaphragm (L) 1 1 1Diaphragm (R) 1 1 1 Latissimus — — 1 Dorsi (L) Latissimus — — 1 Dorsi(R) Pectoralis 1 1 1 Major (p) Pectoralis 1 1 1 Major (d) Tibialis — 1 1Anterior (p) Tibialis — 1 1 Anterior (d) Triceps Brachii 1 1 1 (p)Triceps Brachii 1 1 1 (d) Bicep Femoris 1 1 1 (p) Bicep Femoris 1 1 1(d) Tongue — — — Small Jejunum 1 1 1 Intestine Duodenum — — 1 Heum — — 1Spinal Cervical 1 1 1 Cord Thoracic 1 1 1 Lumbar 1 1 1 Testes Left 1 1 1Right — — 1 ^(a)In the event of insufficient organ/tissue to completeall collections, priority were placed on the primary samples for DNAanalysis ^(b)The standard Bolon et al., 2013 blocking scheme wasfollowed for both hemispheres (2 sets of standard blocks). Only theright hemisphere was processed to slide for microscopic evaluation. (p)= proximal; (d) = distal; (L) = Left side; (R) = right side,

The DNA 1 tissues from Groups 1 through 4 were analyzed for vectorbiodistribution by qPCR. The DNA 2 tissues from Group 5 were analyzedfor vector biodistribution by qPCR.

Tissue samples for RT-qPCR analysis (100 to 180 mg for each tissuesample, when available) to determine gene expression of 4D-310 invarious Cynomolgus monkey tissues were collected and split into 2samples according to Table 17 below:

TABLE 17 Groups 2-4 Group 1 Group 5 Tissue^(a) Region RNA 1 RNA 1 RNA 2(GLA) (GLA) (GFP) Blood Aorta 1 1 1 vessel Carotid 1 1 1 Pulmonary — — 1Artery Brain^(b) Left 1 1 1 Hemisphere Right 1 1 1 Hemisphere Brain Stem1 1 1 Cerebellum 1 1 1 Dorsal Root Thoracic — — — Ganglia All remaining— — — (L) All remaining 1 1 1 (R) Nerve Sciatic Nerve 1 1 1 Heart Left 22 2 Ventricular Free Wall Left Atrium — — — Right Ventricle 1 1 1 RightAtrium — — — Ventricular 1 1 1 Septum Kidney Right Kidney 1 1 1 LeftKidney 1 1 1 Liver Right Lobe 1 1 1 Left Lobe 1 1 1 Lung 2 2 2 SkeletalDeltoid (p) 1 1 1 Muscle Deltoid (d) 1 1 1 Diaphragm (L) 1 1 1 Diaphragm(R) 1 1 1 Latissimus — — 1 Dorsi (L) Latissimus — — 1 Dorsi (R)Pectoralis 1 1 1 Major (p) Pectoralis 1 1 1 Major (d) Tibialis — 1 1Anterior (p) Tibialis — 1 1 Anterior (d) Triceps Brachii 1 1 1 (p)Triceps Brachii 1 1 1 (d) Bicep Femoris 1 1 1 (p) Bicep Femoris 1 1 1(d) Tongue — — 1 Small Jejunum 1 1 1 Intestine Duodenum — — 1 Heum — — 1Spinal Cervical 1 1 1 Cord Thoracic 1 1 1 Lumbar 1 1 1 Testes Left 1 1 —Right — — — ^(a)In the event of insufficient organ/tissue to completeall collections, priority will be placed on the primary samples for IHC,Protein, DNA, and RNA analyses ^(b)The standard Bolon et al., 2013blocking scheme will be followed for both hemispheres (2 sets ofstandard blocks). Only the right hemisphere will be processed to slidefor microscopic evaluation. (p) = proximal; (d) = distal; (L) = Leftside; (R) = right side.

RNA was extracted from the tissue samples, quantified and then analyzedby RT-qPCR for the detection and quantification of RNA sequence specificto 4D-310. Briefly, samples collected for RT-qPCR analysis werecompletely immersed into labeled 2-mL microfuge cryogenic tubesprefilled with 1.5 mL RNALater. Samples were placed on wet ice, storedrefrigerated (2° C. to 8° C.) for at least 24 hours up to 1 week in theRNALater, and then stored frozen (−60° C. to −90° C.) with the RNALaterremoved until gene expression analysis (RT qPCR). All DNA 1 samples fromanimals m Groups 1-4 were shipped on dry ice to Covance Laboratories,Greenfield, Indiana for analysis. All DNA 2 samples from Group 5 weretransferred to the bioanalytical laboratory at the Testing Facility foranalysis.

The RNA 1 tissues from Groups 1 through 4 were analyzed for GLA geneexpression by RT-qPCR. The RNA 2 tissues from Group 5 were analyzed forGFP gene expression by RT-qPCR.

Protein tissue collection—Samples (100 to 180 mg for each tissue sample,when available) were flash frozen in liquid nitrogen (LN2) and storedfrozen (−60° C. to −90° C.). The Protein 1 tissues were analyzed for GLAactivity according to Table 18 below:

TABLE 18 Groups 2-4 Group 1 Group 5 Tissue^(a) Region Protein 1 Protein1 Protein 2 Protein 2 (GLA) (GLA) (GFP) (GFP) Blood Aorta 1 1 1 1 vesselCarotid 1 1 1 1 Pulmonary — — — — Artery Brain^(b) Left 1 1 1 1Hemisphere Right 1 1 1 1 Hemisphere Brain Stem 1 1 1 1 Cerebellum 1 1 11 Nerve Sciatic Nerve 1 1 1 1 Heart Left 2 2 2 2 Ventricular Free WallLeft Atrium 1 1 1 1 Right Ventricle 1 1 1 1 Right Atrium — — — —Ventricular 1 1 Septum Kidney Right Kidney 1 1 1 1 Left Kidney 1 1 1 1Liver Right Lobe 1 1 1 1 Left Lobe 1 1 1 1 Lung 2 2 2 2 Skeletal Deltoid(p) 1 1 1 1 Muscle Deltoid (d) 1 1 1 1 Diaphragm 1 1 1 1 (L) Diaphragm 11 1 1 (R) Latissimus — — 1 1 Dorsi (L) Latissimus — — 1 1 Dorsi (R)Pectoralis 1 1 1 1 Major (p) Pectoralis 1 1 1 1 Major (d) Tibialis — 1 11 Anterior (p) Tibialis — 1 1 1 Anterior (d) Triceps 1 1 1 1 Brachii (p)Triceps 1 1 1 1 Brachii (d) Bicep Femoris 1 1 1 1 (p) Bicep Femoris 1 11 1 (d) Tongue — — 1 1 Small Jejunum 1 1 1 1 Intestine Duodenum — — 1 1Heum — — 1 1 Spinal Cervical 1 1 1 1 Cord Thoracic 1 1 1 1 Lumbar 1 1 11 Testes Left — — — — Right 1 1 — — ^(a)In the event of insufficientorgan/tissue to complete all collections, priority were placed on theprimary samples for IHC, Protein, DNA, and RNA analyses. ^(b)Thestandard Bolon et al., 2013 blocking scheme will be followed for bothhemispheres (2 sets of standard blocks). Only the right hemisphere willbe processed to slide for microscopic evaluation (p) = proximal; (d) =distal; (L) = Left side; (R) = right side

The Protein 2 tissues were analyzed for GFP by ELISA.

For histology and immunohistochemistry (IHC), samples of each specifiedtissue were collected and processed. Samples were fixed in neutralbuffered formalin for 24 to 48 hours, transferred to 70% EtOH as neededfor up to 72 hours, and processed to the block stage for histology andIHC. Efforts were made to preserve the morphology/anatomical integrityof the tissues. A subset of formalin-fixed, paraffin-embedded blocksfrom each designated tissue were shipped to the Sponsor. Slides wereprepared and stained for IHC evaluations.

The Anti-Galactosidase alpha (GLA) Immunohistochemistry (IHC) assay wasperformed using recombinant rabbit anti-human monoclonalAnti-Galactosidase alpha antibody (Abcam clone EP5858, catalog#ab168341) at a final concentration of 10 ug/mL diluted in RocheDiscovery antibody RUO (catalog #05266319001). GLA IHC was performed onthe Roche Discovery ULTRA autostainer platform using Roche DiscoveryChromoMap DAB RUO (catalog #05266645001), Roche Discovery Anti-Rabbit HQRUO (catalog #07017812001) and Roche Discovery Anti-HQ HRP RUO (catalog#07017936001) detection reagents.

GLA IHC assay antigen retrieval was performed at 100° C. for 32 minutesusing Roche Cell Conditioning 1 (CCI) antigen retrieval solution(catalog #950.124). GLA IHC primary antibody and rabbit IgG isotypecontrol antibody (Cell Signaling Technology Rabbit (DA1E) mAb IgG XPIsotype control catalog #13900) were incubated at room temperature for32 minutes. GLA IHC assay secondary antibody and HRP polymer detectionreagents were each incubated at room temperature for 16 minutes. Slideswere counterstained at room temperature using Roche Hematoxylin for 8minutes (catalog #05266726001) and Roche Bluing Reagent for 8 minutes(catalog #05266769001). After removal in the Roche Discovery ULTRAinstrument, slides are rinsed in D1 water with Dawn liquid soap toremove residual oil. GLA IHC slides are then dehydrated through gradedethanols/xylenes and coverslipped using Leica XL and CV5030stainer/coverslipper instruments. GLA IHC slides were then converted tohigh resolution whole slide images using Mikroscan SL2 instrument andassociated Q2 imaging software. GLA IHC slides were evaluated by aboard-codified pathologist. Representative images were captured usingQuPath and ImageScope software.

The following parameters and endpoints were evaluated in this study:modality, clinical signs, body weights, body weight changes,ophthalmology examinations, clinical pathology parameters (hematology,coagulation, and clinical chemistry), gross necropsy findings, organweights, and histopathologic examinations. In addition, DNA (qPCR), RNA(RT-qPCR) and protein (plasma and/or tissue) distribution for enhancedgreen fluorescent protein (EGFP) and alpha-galactosidase A (GLA) wereconducted to evaluate the biodistribution and relative gene expressionefficiency of the test articles Exploratory immunohistochemistry (IHC)evaluations were also conducted.

Results

Mortality 13 all aminals survived to the scheduled necropsy.

Detailed clinical/veterinary observations—There were no clear 4D-310 or4D-C102.CAG-EGFP-related clinical signs.

Body Weight and Body Weight Gains—There were no 4D-310 or4D-C102.CAG-EGFP-related changes in body weight or body weight gain.Slight increases and/or decreases were observed in animals from allgroups during the treatment period and were considered to be withinnormal variation for this species.

Ophthalmology Examinations—There were no 4D-310 or4D-C102.CAG-EGFP-related effects noted in the ophthalmoscopicexaminations. Observations noted were representative of pathology thatwould be expected for animals of this age and species and are notconsidered test article-related.

Hematology—There were no 4D-310 or 4D-C102.CAG-EGFP-related effects onhematology endpoints at any dose level.

Coagulation—There were no apparent 4D-C102.CAG-EGFP-related effects oncoagulation endpoints.

qPCR GLA (DNA)_Biodistribution Analysis

The quantity of 4D-310 vector DNA present in each sample was calculatedfrom the average of the two unspiked replicates analyzed with qPCR.Results are reported as copies of 4D-310 present per μg of DNA analyzed(adjusted for any dilution factors and concentrations below 0.2 μg/μL).Group 1 is a vehicle/control group. All the tested samples from thevehicle treated animal (Animal ID 1001) were found to be <LOD (limit ofdetection) for GLA DNA. Among animals administered 3.0×10¹² vg/kg4D-310, GLA DNA was detected positive, ranging from 51.72 (Jejunum ofAnimal 2003) to 6,309,088.75 (Liver, Right Lobe of Animal 2002) copiesper μg of total DNA for all tested tissue samples except for the samplesmentioned below. Testes, Left of Animals 2001 and 2003, and Spinal Cord,Lumber of Animals 2002 and 2003 were found to be <LOQ. No result toreport for Heart, Left Atrium of Animal 2002 due to no remaining tissueavailable. Result for Carotid of Animal 2002 was obtained by analyzingthe DNA from the failed extraction run upon sponsor's request. Amonganimals administered 1.0×10¹³ vg/kg 4D-310, GLA DNA was found aspositive in all tested tissue samples. The testing results obtainedrange from 178.65 (Jejunum of Animal 3002) to 3,379,109.30 (Liver, LeftLobe of Animal 3001) copies per μg of total DNA. The original testingresults of Liver, Right Lobe and Liver, Left Lobe of Animals 3002 and3003 were found to be >ULOQ (upper limit of quantification), DNAextracted from these tissue samples were retested with further 1:10dilution performed.

Among animals administered 5.0×10¹¹ vg/kg 4D-310, GLA DNA was found aspositive in all tested tissue samples. The testing results obtainedrange from 168.86 (Testes of Animal 4003) to 21,116,311.46 (Liver, RightLobe of Animal 4001) copies per μg of total DNA. The original testingresults of Liver, Right Lobe and Liver Left Lobe of Animals 4001, 4002and 4003 were all found to be >ULOQ. DNA extracted from these tissuesamples were retested with further 1:50 (for 4001), 1:20 (for 4002) and1:25 (for 4003) dilution performed.

qPCR distribution of 4D-310 to key Fabry tissues following IVadministration of 3×10¹² vg/kg to the non-human primates is shown atFIG. 15 , with dose responsive biodistribution to the liver achieved.These data demonstrate that 4D-310 delivered IV successfully localizesto key tissues of Fabry disease pathology, including the heart andkidneys. These data are also supportive of dose responsive expression inthe liver as a target for systemic expression of soluble therapeuticprotein.

GLA Plasma Quantification

A total of 50 plasma samples from animals administered 3.0×10¹² to5.0×10¹³ vg/kg 4D-310 on Days 1, (predose), 15, 29, 43 and the day ofnecropsy were analyzed for GLA activity using a qualified method. Theresults from calibration standards and quality control samplesdemonstrated acceptable performance of the method for al reportedconcentrations.

GLA levels were comparable across all animals prior to dosing (Day 1).By Day 15, plasma GLA levels increased in 1 of 3 animals at 3.0×10¹²vg/kg 4D-310 (up to 23.6×), 3 of 3 animals at 1.0×10¹³ vg/kg 4D-310 (upto 8.1×) and 3 of 3 animals at 5.0×10¹³ vg/kg 4D-310 (up to 152.2×).Plasma GLA levels were generally similar (within approximately 2.5-foldof the Day 15 value) from Day 15 through the day of necropsy. This datais summarized below:

4D-310 Dose Level Fold-change from Group (vg/kg) Day control 2 3.0 ×10¹²  1 (predose) — 15 23.6x 29 25.6x 43 42.3x Day of necropsy 20.9x 1.0× 10¹³  1 (predose) — 15 5.2-8.1x 29 2.0-5.6x 43 2.5-5.3x Day ofnecropsy 2.3-6.3x 5.0 × 10¹³  1 (predose) — 15 26.5-152.2x 29 19.2-65.7x43 15.9-70.9x Day of necropsy 19.7-73.4x

FIG. 16 depicts AGA in NHP plasma after a single intravenous dose of4D-310, grouped by dose (a 54.7-fold increase in plasma AGA level wasobserved in the 5.0×10¹³ vg/kg dose relative to vehicle-treatedcontrol). High AGA level in animal #2002 caused the low dose group meanto be higher than the mid dose group mean. Otherwise a dose-dependencywas observed. Delivery of 4D-310 at all dose groups evaluated resultedin plasma AGA levels above endogenous AGA levels (vehicle), whichdemonstrates successful gene delivery and gene expression from the4D-310 product.

FIG. 17 depicts plasma AGA levels in individual animals after a singleintravenous dose of 4D-310. Animal 2002 has unexpectedly high plasma AGAactivity—this animal has high DNA, RNA, and protein in the liver.Delivery of 4D-310 resulted in plasma AGA levels above endogenous AGAlevels (vehicle) for all individual animals, which demonstratessuccessful gene delivery and gene expression from the 4D-310 product.

GLA Protein Tissue Biodistribution Analysis

The Anti-Galactosidase alpha (GLA) Immunohistochemistry (IHC) assay wasdeveloped by screening multiple commercially available GLA antibodieswith a matrix of assay conditions including antigen retrievalconditions, primary antibody and detection conditions in formalin fixedparaffin embedded (FFPE) cell pellets and tissues. Specificity andsensitivity of GLA IHC assay was determined by evaluation of wild typeHEK293T and 4D-310 transfected HEK293T cells and 4D-310 treated Fabrymouse tissues.

4D-310 treated formalin fixed paraffin embedded (FFPE) non-human primate(NHP) tissue samples were evaluated for GLA IHC expression. Control cellpellets were included in each experimental run for quality control andto ensure consistent results between runs. Rabbit IgG Isotype antibodycontrols were included for each tissue sample to ensure specificity ofGLA IHC assay.

Low level endogenous GLA expression was observed in vehicle treatedtissues. An increase in GLA IHC expression was observed in 4D-310treated NHP cardiac tissues at all dose levels (3×10¹², 1×10¹³ and5×10¹³). An increase in GLA expression was observed in 4D-310 treatedNHP liver tissues only at 5×10¹³ dose level. Detection at the lower doselevels by IHC was potentially confounded by high endogenous liverexpression, based on the expression seen in the vehicle-treated animal.No increase in GLA IHC staining was observed in NHP skeletal muscle,diaphragm, kidney, aorta, carotid artery or small intestine tissuesamples. Results were verified and cell types identified by an external,board-certified pathologist.

At study termination in animals administered 4D-310, increases in GLAexpression to the vehicle control were limited to the liver, carotidartery, heart (including left ventricular free wall and left atrium),lung, aorta, a subset of skeletal muscles (including deltoid, pectoralismajor and diaphragm), kidney and brain (left hemisphere). The greatestexpression of GLA were seen in the liver (up to 21.1×), carotid artery(up to 15.5×), left ventricular free wall (up to 12.2×), and lung (up to10.1×). A lower level of GLA expression (<10×) was noted in the aorta(up to 7.4×), deltoid (up to 4.3×) diaphragm (up to 3.7×), pectoralismajor (up to 3.4×), kidney (2.5×), bicep femoris (2.5×), brain (up to2.4×), left atrium (2.2×). Elevation in GLA was occasionally, though notalways, dose responsive. These changes were considered 4D-310-related.All other tissues were less than 2× of control and therefore were notconsidered to have elevated GLA expression. A summary of relative GLAtissue expression from animals administered 4D-310 can be found at Table19 below:

TABLE 19 Incidence by Dose Level (fold-change from control) Tissue 3.0 ×10¹² vg/kg 1.0 × 10¹³ vg/kg 5.0 × 10¹³ vg/kg Liver 3 of 3 (3.0-11.5x) 3of 3 (2.0-7.2x) 3 of 3 (4.6-21.1) Carotid 0 of 3 2 of 3 (3.9-15.5x) 1 of3 (4.2x) Left ventricular 1 of 3 (2.0x) 1 of 3 (2.0-3.2x) 3 of 3(2.0-12.2x) free wall (a + b) Lung 0 of 3 1 of 3 (3.7x) 2 of 3(2.5-10.1x) Aorta 1 of 3 (2x) 3 of 3 (2.3-7.4x) 2 of 3 (2.0-2.4x)Deltoid 0 of 3 2 of 3 (2.1-4.3x) 0 of 3 (proximal and distal) Diaphragm(left 3 of 3 (2.0-3.7x) 2 of 3 (2.3-2.8x) 3 of 3 (2.2-3.6x) and right)Pectoralis Major 2 of 3 (2.0-3.4x) 2 of 3 (2.2-3.0x) 3 of 3 (2.1-2.6x)(proximal and distal) Bicep femoris 0 of 3 0 of 3 1 of 3 (2.5x) Kidney 0of 3 0 of 3 1 of 3 (2.5x) Left atrium 0 of 3 0 of 3 1 of 3 (2.2x) Brain(left and 0 of 3 1 of 3 (2.4x) 1 of 3 (2.1x) right bemisphere)

As can be seen from FIGS. 18-19 , intravenous delivery of 4D-310 to NHPsresulted in a significant increase m AGA expression in Fabry-relevanttissues compared to control NHPs treated with vehicle only. Delivery of4D-310 thus resulted in successful gene delivery and gene expressionfrom the 4D-310 product in therapeutically relevant tissues. ElevatedAGA expression is shown in the heart n the high dose animals (FIG. 19 ).

RT-qPCR (RNA) Gene Expression Analysis

All samples tested from Group 1 (vehicle/control) animal were found tobe <LOD for 4D-310 as expected.

Among the Group 2 animals (that received 3.0×10¹² vg/kg of 4D-310),4D-310 RNA was detected positive, ranging from 630.68 (Testes, Left ofAnimal 2001) to 19,744,452.23 (Liver, Left Lobe of Animal 2002) copiesper □g of total RNA for all tested tissue samples except for thefollowing samples: (1) Deltoid Distal, Diaphragm Left, Diaphragm right,Pectoralis Major and Biceps Femoris Distal and Brain Cerebellum,Jejunum, Carotid, Spinal Cord Thoracic, Spinal Cord Lumbar and Lung ofAnimal 2001 were found to be <LOD (2) Brain, Brainstem of Animal 2001was found to be <LOQ (3) Deltoid Distal and Biceps Femoris Distal ofAnimals 2002 were found to be <LOD (4) Deltoid Proximal, PectoralisMajor Proximal, Biceps Femoris Proximal, Jejunum, DRG All remainingRight and Spinal Cord Thoracic of Animals 2002 were found to be <LOQ (5)Diaphragm Left, Diaphragm Right, Pectoralis Major Proximal, PectoralisMajor Distal, Biceps Femoris Distal, Sciatic Nerve and Spinal CordLumber of Animal 2003 were found to be <LOD (6) Deltoid Proximal, BicepsFemoris Proximal, Brain Right Hemisphere, Brain Cerebellum, DRG AUremaining Right and Testes Left of Animal 2003 were found to be <LOQ. Nocopies per μg of total RNA result to report for Sciatic Nerve and SpinalCord Lumbar of Animal 2002 due to the negative Total RNA concentrationmeasured for these samples.

Among the Group 3 animals (that received 1.0×10¹³ vg/kg of 4D-310),4D-310 RNA was found as positive in all tested tissue samples. Thetesting results obtained range from 765.27 (Jejunum of Animal 3003) to10,387,886.08 (Aorta of Animal 3002) copies per □g of total RNA of alltested tissue samples except for the following: (I) Deltoid Proximal,Deltoid Distal, Diaphragm Left, Pectoralis Major Proximal, PectoralisMajor Distal, Triceps Branchii Proximal, Triceps Branchii Distal, BicepsFemoris Proximal, Biceps Femoris Distal, Sciatic Nerve and Spinal CordLumbar of Animal 3001 were found to be <LOD (2) Diaphragm Right ofAnimal 3001 had no copies/μg of RNA result available (3) Brain,Brainstem and Lung of Animal 3001 were found to be <LOQ (4) DiaphragmRight, Pectoralis Major Proximal, Jejunum, Sciatic Nerve, Lung andSpinal Cord Lumbar of Animal 3002 were found to be <LOD (5) BicepsFemoris Distal and Bran Brainstem of Animal 3002 were found to be <LOQ(6) DRG All remaining Right of Animal 3003 was found to be <LOQ.

Among the Group 4 animals (that received 5.0×10¹³ vg/kg of 4D-310),4D-310 RNA was found as positive in all tested tissue samples. Thetesting results obtained range from 930.51 (Triceps Branchii, Proximalof Animal 4001) to 127,602,838.22 (Heart, Ventricular Septum of Animal4002) copies per μg of total RNA except for the following: (1) BrainCerebellum, Jejunum and Sciatic Nerve of Animal 4001 were found to be<LOD (2) Carotid for Animal 4001 was found to be <LOQ (3) Brain LeftHemisphere, Brain Right Hemisphere, Brain Brainstem, DRG All remainingRight and Lung of Animal 4001 had negative total RNA concentration andtherefore no copies/μg RNA result is available for these samples (4)Diaphragm Left and Biceps Femoris Proximal of Animal 4002 were found tobe <LOD (5) Jejunum and Sciatic Nerve of Animal 4002 were found to be<LOQ (6) Biceps Femoris Distal and Sciatic Nerve of Animal 4003 werefound to be <LOD (7) Deltoid Proximal, Pectoralis Major Proximal andJejunum of Animal 4003 were found to be <LOQ.

The bridging assessment to identify differences, if any, between the useof manually purified RNA samples and those purified using the QIAcube HTsystem demonstrated that both methods yielded similar results with noeffect on the precision or accuracy of the method. The inter-assayprecision between the different QC types, yielded Qty % CV valuesbetween 2% and 20% for all QC levels, while the inter-assay accuracy Qty% RE results ranged from −6% to −2% across all QC concentrations.

Approximately 52 days following intravenous infusion, the tissuesexhibiting the highest levels of vector derived gene expression were theleft ventricular free wall and ventricular septum heart samples, theleft and right liver lobes, the carotid blood vessel, and rightventricle heart samples. The left ventricular free wall and ventricularseptum heart samples yielded an average of 5.48×10³ and 5.46×10³ copiesper 10 ng of total RNA analyzed, while the average concentration acrossall 6 liver samples was 3.59×10³ copies. The carotid blood vessel andright ventricle heart samples yielded 3.33×10³ and 1.78×10³ copiesrespectively. A majority of the remaining tissue samples yielded minimalto no vector derived gene expression with more than ¾ of the samplestesting at or below the limit or quantitation. Stark contrasts areevident when comparing the biodistribution results, which test just forthe presence of the viral vector, with the gene expression results. Fromthe biodistribution data, the levels of vector detected in the liversamples were mon than 60× greater at 5.75×10⁶ copies per μg of sampleDNA compared to 8.69×10⁴ copies for the left ventricular free wallsamples and 9.12×10⁴ copies from the ventricular septum samples.Conversely, the levels of gene expression observed were approximately1.5× higher in the 2 heart samples compared to the liver samples. Thecarotid blood vessel and right ventricle heart samples showed similarresults with vector levels ˜128× and ˜69× higher in the liver samples,while the gene expression levels were approximately equal between theblood vessel and liver samples and only 2× higher in the liver samplesversus the right ventricle heart samples. Vector DNA was detected in theuntranscribed liver samples of all animals. This contamination of thevector DNA would account for approximately 1% to 12% of the targetsequence gene expression. Vector contamination was not detected in anyof the remaining samples analyzed. The expression levels of thehousekeeping gene, Hprt1, was consistent across the individual tissuesof each animal confirming the overall integrity of the RNA samples.

As can be seen from FIG. 20 , a single intravenous administration ofeach of the specified doses of 4D-310 to NHPs results in GLA RNAexpression with consistent expression observed in the heart and liver,both key organs for treating Fabry disease.

Gross Pathology

There were no 4D-310 or 4D-C102.CAG-EGFP-related macroscopic findings.Macroscopic findings in all test article animals given 4D-C102 CAG-EGFPat 5.0×10¹³ and the single vehicle control animal consisted of whiteforeign material in the quadriceps (bilateral or unilateral). One of theanimals (Animal No. 5003) given 41-C102.CAG-EGFP also had a unilateralabscess in the quadriceps. These were presumably procedure related asthey occurred in the control and 4D-C102.CAG-EGFP-treated animals. Oneanimal given the low dose of 4D-310 at 3.0×10¹² (Animal No 2002) hadedema of the subcutis in the right hindleg/limb. The later change waslikely not directly test article related as it was a unilaterallocalized change. Macroscopic changes were not evaluated microscopicallyas per protocol.

Histopathology

There were no clear 4D-310 or 4D-C102.CAG-EGFP-related microscopicfindings. Several findings occurred in tissues of animals with no cleartest article relationship. Findings noted in the 4D-310 and4D-C102.CAG-EGFP-treated animals and the vehicle control consisted ofminimal infiltrates of mononuclear cells in adventitia of varioustissues including heart, various skeletal muscles, and sciatic nerve.Additionally, minimal mononuclear infiltrates occurred in the meningesof the brain of 4D-310-treated animals, but not in 4D-C102.CAG-EGFPtreated or the vehicle control animals. Other findings noted in 4D-310treated monkeys included minimal myofiber degeneration or regenerationin various skeletal muscles (typically focal), minimal alveolarmacrophage aggregates in the lung, and minimal (multifocal) vacuolationof hepatocytes. These were typically of low incidence with no doserelated trend in incidence, distribution, or severity. Furthermore,these minimal changes can occur as background findings for the speciesand thus were not considered test article related. None of thesefindings are considered adverse. Fat necrosis occurred in the adiposetissue of various tissues of 4D-310-treated animals including the heartcoronary groove, aorta adventitia, and skeletal muscle adventitia withno clear dose relationship in incidence or severity. Severity wasminimal to moderate and was not dose related. Fat necrosis has beennoted as a consequence of high dose methylprednisolone treatment in ourlaboratory or can be noted as an incidental background change.Methylprednisolone treatment was used to control immunogenic response ofthe test articles.

Autophagy of nerve cell bodies (cytoplasmic rarefaction) of scatteredneurons was noted in the dorsal root ganglia of one animal at each doseof 4D-310. Severity was minimal to mild but was not dose related. Thischange is a background finding in Cynomolgus monkeys (Butt M. et al.,2019)(Pardo I. et al., 2019) and is not considered test article related.All other microscopic observations were considered incidental and nottest article related. These observations are known background findingsfor the species, were of low incidence, were of similar incidence andseverity for control and test article-treated animals, and/or had nodose response relationship.

Toxicology—Changes related to IV administration of 4D-310 or4D-C102.CAG-EGFP were limited to effects on clinical pathology.Following a single IV dose of 5.0×10¹³ vg/kg 4D-310 and4D-C102.CAG-EGFP, there were minimal to marked, respectively, increasesin alanine aminotransferase (ALT) activities. In animals administered4D-C102.CAG-EGFP, there were concurrent minimal to moderate increases inaspartate aminotransferase (AST) activities, gamma-glutamyltransferase(GGT) activities, and total bilirubin concentration. These changes weremost pronounced on Day 14 and partially to fully resolved at laterintervals. These changes were considered 4D-310 and4D-C102.CAG-EGFP-related, and were indicative of hepatocellular and/orhepatobiliary effects. No microscopic correlates were observed attermination.

Also at 5.0×10¹³ vg/kg 4D-C102.CAG-EGFP, there was a transient minimaldecrease in mean urea nitrogen concentration on Day 21, and minimal tomild decreases in albumin concentrations beginning on Day 35, which wereconsidered 4D-C102.CAG-EGFP-elated. The hepatocellular effects discussedabove may have contributed to these changes. At ≥3.0×10¹² vg/kg 4D-310them were transient minimal to mild increases in creatine kinase (CK)activities on Day 7, which were considered 4D-310-related and indicativeof a minor muscle effect. No microscopic correlates were observed attermination.

GLA Pharmacodynamics and Biodistribution—Circulating (plasma) GLAprotein was elevated in a somewhat dose responsive manner beginning onDay 15 (the first post dose timepoint tested) through termination. ByDay 15, plasma GLA levels increased in 1 of 3 animals at 3.0×10¹² vg/kg4D-310 (up to 23.6×), 3 of 3 animals at 1.0×10¹³ vg/kg 4D-310 (up to8.1×) and 3 of 3 animals at 5.0×10¹² vg/kg 4D-310 (up to 152.2×). PlasmaGLA levels were generally similar (within approximately 2.5-fold of theDay 15 value) from Day 15 through the day of necropsy. At studytermination in animals administered 4D-310, GLA DNA was found in mosttissues tested in a somewhat dose responsive manner. In animalsadministered 3.0×10¹² vg/kg 4D-310, vector copies/ug of DNA ranged from51.72 to 6.3×10⁶ with a small subset of tissues having undetectablelevels of GLA DNA. At 1.0×10¹³ vg/kg 4D-310, GLA DNA was found in alltissues assayed with copies per μg of DNA ranging from 178 to 3.4×10⁶.Similarly, at 5.0×10¹³ vg/kg 4D-310, GLA DNA was detectable in alltissues assayed with copies per ug of DNA ranging from 168 to 21.1×10⁶.

Translation was confirmed by detectable GLA protein in a subset oftissues including liver, carotid artery, heart (including leftventricular free wall and left atrium), lung, aorta, a subset ofskeletal muscles (deltoid, pectoral, major and diaphragm), kidney andbrain (left hemisphere). See FIGS. 21A-B and 22A-B. The greatestexpression of GLA was seen in the liver (up to 21.1×), carotid artery(up to 15.5×), left ventricular free wall (up to 12.2×), and lung (up to10.1×). A lower level of GLA expression (<10×) was noted in the aorta(up to 7.4×), deltoid (up to 4.3×) diaphragm (up to 3.7×), pectoralismajor (up to 3.4×), kidney (2.5×), bicep femoris (2.5×), brain (up to2.4×), left atrium (2.2×). Elevation in GLA was occasionally, though notalways, dose responsive.

EGFP Pharmacodynamics and Biodistribution—At study termination inanimals administered 5.0×10¹³ vg/kg 4D-C102.CAG.EGFP, EGFP DNA was foundin most tissues tested, the tissues exhibiting the highest levels ofvector derived gene expression were the left ventricular free wall andventricular septum (heart), the left and right liver lobes, the carotidblood vessel, and right ventricle (heart). The levels of gene expression(RNA) observed were approximately 1.5× higher in the 2 heart samplescompared to the liver samples. The carotid blood vessel and rightventricle heart samples showed similar results with DNA vector levels˜128× and ˜69× higher in the liver samples, while the gene expressionlevels were approximately equal between the blood vessel and liversamples and only 2× higher in the liver samples versus the rightventricle heart samples. Translation of the GFP protein was observed inmost tissues assayed and generally aligned with the biodistribution andgene expression data. The greatest expression (marked increases) of GFPwas seen in the heart (up to 8,956,503×), blood vessels (up to 22,991×),and liver (up to 5,194×). Moderate GFP expression was noted in thekidney, deltoid and diaphragm muscles, up 247×, 989× and 347×,respectively. There was minimal expression observed in the brain, nerve,tongue, spinal cord, bicep and tricep muscles. Taken together, a singleIV infusion of 4D-310 or 4D-C102.CAG-EGFP was well tolerated in malecynomolgus monkeys up to 5.0×10¹³ vg/kg (the highest dose tested for4D-310 and the only dose tested for 4D-C102.CAG-EGFP) with measurablevector DNA, gene expression (RNA and protein) in various tissues mostnotably the liver, heart and blood vessels. Due to the lack of anyadverse changes, the 5.0×10¹³ vg/kg dose level was determined to be theno-observed-adverse-effect level (NOAEL) for both 4D-310 or41-C102.CAG-EGFP under the conditions of this study.

CONCLUSION

A single intravenous infusion of 4D-310 or 4D-C102.CAG EGFP was welltolerated in male cynomolgus monkeys up to 5.0×10¹³ vg/kg (the highestdose tested for 4D-310 and the only dose tested for 4D-C102.CAG-EGFP)with measurable vector DNA, gene expression (RNA and protein) in varioustissues most notably in the liver, heart and blood vessels. Due to thelack of any adverse changes, the 5.0×10¹³ vg/kg dose level wasdetermined to be the no-observed-adverse-effect level (NOAEL) for both4D-310 and 4D-C102.CAG.EGFP under the conditions of this study.

Vector genomes, RNA message and functional protein were observed in theheart, especially at the high (5×10¹³ vg/kg) dose. Vector genomes andRNA message appeared to correlate. RNA message and AGA activity alsoappeared to correlate. AGA activity data in Fabry-relevant tissues wasconfirmed by IHC. Measured levels of RNA message and protein with EGFPwere lower than those measured with GLA

While the materials and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the method described hereinwithout departing from the concept, spirit and scope of the invention.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention.

1. A method for delivering a heterologous nucleic acid comprising anucleotide sequence encoding a gene product to a kidney cell, braincell, and/or lung cell, the method comprising contacting the kidneycell, brain cell and/or lung cell with a recombinant adeno-associatedvirus (rAAV) comprising: (a) a variant AAV capsid protein comprising aheterologous peptide insertion with a length of 7 to 20 amino acidscovalently inserted in the GH-loop of the capsid protein relative to acorresponding parental AAV capsid protein, wherein the peptide insertioncomprises the amino acid sequence NKTTNKD and (ii) a heterologousnucleic acid comprising a nucleotide sequence encoding a gene product,said nucleotide sequence operably linked to a promoter.
 2. The methodaccording to claim 1, wherein the insertion peptide has from 1 to 3spacer amino acids (Y₁-Y₃) at the amino and/or carboxyl terminus ofamino acid sequence NKTTNKD.
 3. The method according to claim 2, whereinthe insertion peptide is LANKTTNKDA.
 4. The method according to claim 1,wherein the variant capsid protein comprises a V708I amino acidsubstitution relative to VP1 of AAV2 (SEQ ID NO:2) or the correspondingposition in the capsid protein of another AAV serotype and wherein thevariant capsid protein comprises an amino acid sequence at least 90%identical to the entire length of the amino acid sequence set forth asSEQ ID NO:4.
 5. The method according to claim 4, wherein the variantcapsid protein comprises an amino acid sequence at least 95% identicalto the entire length of the amino acid sequence set forth as SEQ IDNO:4.
 6. The method according to claim 1, wherein the insertion site islocated between two adjacent amino acids at a position between aminoacids 570 and 611 of VP1 of AAV2, or the corresponding position in thecapsid protein of another AAV serotype.
 7. The method according to claim6, wherein the insertion site is located between amino acidscorresponding to amino acids 587 and 588 of VP1 of AAV2 or between aminoacids corresponding to amino acids 588 and 589 of VP1 of AAV2 or thecorresponding position in the capsid protein of another AAV serotype. 8.The method according to claim 1, wherein the promoter is a constitutivepromoter.
 9. The method according to claim 1, wherein the promoter is atissue-specific promoter.
 10. A method for delivering a heterologousnucleic acid comprising a nucleotide sequence encoding a gee product tothe kidney, brain, spinal cord or lung of a primate, the methodcomprising administering to the primate a recombinant adeno-associatedvims (rAAV) comprising: (a) a variant AAV capsid protein comprising aheterologous peptide insertion with a length of 7 to 20 amino acidscovalently inserted in the GH-loop of the capsid protein relative to acorresponding parental AAV capsid protein, wherein the peptide insertioncomprises the amino acid sequence NKTTNKD and (ii) a heterologousnucleic acid comprising a nucleotide sequence encoding a gene product,said nucleotide sequence operably linked to a promoter, or administeringto the subject a pharmaceutical composition comprising the rAAV and apharmaceutically acceptable excipient.
 11. The method according to claim10, wherein the insertion peptide has from 1 to 3 spacer amino acids(Y₁-Y₃) at the amino and/or carboxyl terminus of amino acid sequenceNKTTNKD.
 12. The method according to claim 11, wherein the insertionpeptide is LANKTTNKDA.
 13. The method according to claim 10, wherein thevariant capsid protein comprises a V708I amino acid substitutionrelative to VP1 of AAV2 (SEQ ID NO:2) or the corresponding position inthe capsid protein of another AAV serotype and wherein the variantcapsid protein comprises an amino acid sequence at least 90% identicalto the entire length of the amino acid sequence set forth as SEQ IDNO:4.
 14. The method according to claim 13, wherein the variant capsidprotein comprises an amino acid sequence at least 95% identical to theentire length of the amino acid sequence set forth as SEQ ID NO:4. 15.The method according to claim 10, wherein the insertion site is locatedbetween two adjacent amino acids at a position between amino acids 570and 611 of VP1 of AAV2, or the corresponding position in the capsidprotein of another AAV serotype.
 16. The method according to claim 15,wherein the insertion site is located between amino acids correspondingto amino acids 587 and 588 of VP1 of AAV2 or between amino acidscorresponding to amino acids 588 and 589 of VP1 of AAV2 or thecorresponding position in the capsid protein of another AAV serotype.17. The method according to claim 10, wherein the promoter is aconstitutive promoter.
 18. The method according to claim 10, wherein thepromoter is a tissue-specific promoter.
 19. The method according toclaim 10, wherein the rAAV or pharmaceutical composition is administeredto the primate by intravenous injection.
 20. The method according toclaim 10, wherein the primate is a human.